Intra-assay Accuracy Research Articles

Qualification of a select one‐stage activated partial thromboplastin time‐based clotting assay and two chromogenic assays for the post‐administration monitoring of nonacog beta pegol

Background Nonacog beta pegol (N9-GP) is an extended half-life, glycoPEGylated recombinant human factorIX that is under development for the prophylaxis and treatment of bleeding episodes in hemophiliaB patients. Considerable reagent-dependent variability has been observed when one-stage clotting assays are used to measure the recovery of recombinant FIX products, including N9-GP. Objective To qualify select one-stage clotting and chromogenic FIX activity assays for measuring N9-GP recovery. Methods The accuracy and precision of the one-stage clotting assay (with the STA-Cephascreen activated partial thromboplastin [APTT] reagent) and the ROX FactorIX and BIOPHEN FactorIX chromogenic assays for measuring N9-GP recovery were assessed in N9-GP-spiked hemophilia B plasma samples in a systematic manner at three independent sites, with manufacturer-recommended protocols and/or site-specific assay setups, including different instruments. Results For each of the three FIX activity assays qualified on five different reagent-instrument systems, acceptable intra-assay and interassay accuracy and precision, dilution integrity, reagent robustness and freeze-thaw and short-term sample stabilities were demonstrated. The STA-Cephascreen assay showed a limited reportable range at one of the three qualification sites, and the BIOPHEN FactorIX assay showed suspect low-end sensitivity at one of the three qualification sites. An individual laboratory would account for these limitations by adjusting the assay's reportable range; thus, these findings are not considered to impact the respective assay qualifications. Conclusion The one-stage clotting assay with the STA-Cephascreen APTT reagent, the ROX FactorIX chromogenic assay and the BIOPHEN FactorIX chromogenic assay are considered to be qualified for the measurement of N9-GP in 3.2% (0.109m) citrated human plasma.

Simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum using an immobilized trypsin

Proteomic approaches using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) without an immunopurification technique have not been applied to the determination of serum cetuximab. This study developed a simple and rapid LC-MS/MS method for the absolute determination of cetuximab in human serum and applied it to clinical settings. Surrogate peptides derived from cetuximab digests were selected using a Fourier transform mass spectrometer. Reduced-alkylated serum cetuximab without immunopurification was digested for 20minutes using immobilized trypsin, and the digestion products were purified by solid-phase extraction. The LC-MS/MS was run in positive ion multiple reaction monitoring mode. This method was applied to the determination of serum samples in head and neck cancer patients treated with cetuximab. The chromatographic run time was 10minutes and no peaks interfering with surrogate peptides in serum digestion products were observed. The calibration curve of absolute cetuximab in serum was linear over the concentration range of 4–200μg/mL. The lower limit of quantification of cetuximab in human serum was 4μg/mL. The intra-assay and inter-assay precision and accuracy were less than 13.2% and 88.0–100.7%, respectively. The serum concentration range of cetuximab was 19–140μg/mL in patients. The serum cetuximab concentrations in LC-MS/MS were correlated with those in ELISA (r=0.899, P <0.01) and the mean bias was 1.5% in cancer patients. In conclusion, the present simple and rapid method with acceptable analytical performance can be helpful for evaluating the absolute concentration of serum cetuximab in clinical settings.

Cathepsin-S degraded decorin are elevated in fibrotic lung disorders \u2013 development and biological validation of a new serum biomarker

BackgroundDecorin is one of the most abundant proteoglycans of the extracellular matrix and is mainly secreted and deposited in the interstitial matrix by fibroblasts where it plays an important role in collagen turnover and tissue homeostasis. Degradation of decorin might disturb normal tissue homeostasis contributing to extracellular matrix remodeling diseases. Here, we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) quantifying a specific fragment of degraded decorin, which has potential as a novel non-invasive serum biomarker for fibrotic lung disorders.MethodsA fragment of decorin cleaved in vitro using human articular cartilage was identified by mass-spectrometry (MS/MS). Monoclonal antibodies were raised against the neo-epitope of the cleaved decorin fragment and a competitive ELISA assay (DCN-CS) was developed. The assay was evaluated by determining the inter- and intra-assay precision, dilution recovery, accuracy, analyte stability and interference. Serum levels were assessed in lung cancer patients, patients with idiopathic pulmonary fibrosis (IPF), patients with chronic obstructive pulmonary disease (COPD) and healthy controls.ResultsThe DCN-CS ELISA was technically robust and was specific for decorin cleaved by cathepsin-S. DCN-CS was elevated in lung cancer patients (p < 0.0001) and IPF patients (p < 0.001) when compared to healthy controls. The diagnostic power for differentiating lung cancer patients and IPF patients from healthy controls was 0.96 and 0.77, respectively.ConclusionCathepsin-S degraded decorin could be quantified in serum using the DCN-CS competitive ELISA. The clinical data indicated that degradation of decorin by cathepsin-S is an important part of the pathology of lung cancer and IPF.

GC-MS Method for Quantification of Mephedrone in Human Blood Sample.

Abuse of mephedrone (4-methylmethcathinone) has emerged among the last few years. Intoxication and fatality associated with mephedrone have been reported in the literature. The present work describes a simple and rapid gas chromatography-mass spectrometry (GC-MS) method for detection and quantification of mephedrone in human blood samples using GC-MS. Blood was deproteinated with acetonitrile and the supernatant was derivatized and extracted in a single step with ethyl chloroformate and ethyl acetate. Methamphetamine-d5 was used as an internal standard (IS). The characteristic ions at m/z 58, 119 and 130 for mephedrone and m/z 62, 92 and 134 for methamphetamine-D5 were used for quantification of the analyte. The calibration curve was linear within the concentration range 10-2,000 ng/mL. Intra- and inter-assay precisions (%RSD) were within 10.9-11.9 and 9.2-11.2, respectively. Intra-assay accuracy (%Bias) was between 1.8 and 2.8. The method was successfully applied to detect and quantify mephedrone in real blood samples.

An ultra-high-pressure liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the detection of cannabinoids in whole blood using solid phase extraction

ABSTRACTA novel high-throughput method using automated solid-phase extraction (SPE) coupled with ultra-high-pressure liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was developed in order to quantify Δ9-tetrahydrocannabinol (THC) and its major metabolites and screen for several synthetic cannabinoids. Cannabinoids and synthetic cannabinoids were extracted from ovine whole blood using Phenomenex Strata-X Drug B SPE columns. Multiple reaction monitoring (MRM) mode was used to monitor at least two transitions of the analyte and its deuterated internal standard. Calibration was fitted quadratically (R2 > 0.995) over a range from 1 to 20 ng/mL for THC, 11-hydroxy-THC, cannabinol and cannabidiol, and 10 to 200 ng/mL for 11-nor-Δ9-carboxy-tetrahydrocannabinol and its glucuronide. Inter-assay accuracy and precision were evaluated over n = 3 analyses spanning six days. With the exception of cannabidiol, the accuracies ranged from 0.52% to 8.63% and the coefficient of variation (%CV) was found to range from 1.91% to 7.69%. Intra-assay accuracy and the precision of these analytes (n = 16) was found to range from 0.16% to 11.42% and from 1.10% to 8.40%, respectively. Cannabidiol intra- and inter-assay accuracy and precision were more variable than for other analytes. The procedure minimized specimen handling, extraction and reduced runtime as compared with an existing gas chromatography–mass spectrometry method and permitted the qualitative identification of several synthetic cannabinoid species.

Determination of ∆-9-Tetrahydrocannabinol (THC), 11-hydroxy-THC, 11-nor-9-carboxy-THC and Cannabidiol in Human Plasma using Gas Chromatography-Tandem Mass Spectrometry.

Two marijuana compounds of particular medical interest are delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). A gas chromatography-tandem mass spectrometry (GC-MS-MS) method was developed to test for CBD, THC, hydroxy-THC (OH-THC) and carboxy-THC (COOH-THC) in human plasma. Calibrators (THC and OH-THC, 0.1 to 100; CBD, 0.25 to 100; COOH-THC, 0.5-500 ng/mL) and controls (0.3, 5 and 80 ng/mL, except COOH-THC at 1.5, 25 and 400 ng/mL) were prepared in blank matrix. Deuterated (d3) internal standards were added to 1-mL samples. Preparation involved acetonitrile precipitation, liquid-liquid extraction (hexane:ethyl acetate, 9:1), and MSTFA derivatization. An Agilent 7890 A GC was interfaced with an Agilent 7000 MS Triple Quadrupole. Selected reaction monitoring was employed. Blood samples were provided from a marijuana smoking study (two participants) and a CBD ingestion study (eight participants). Three analytes with the same transitions (THC, OH-THC and COOH-THC) were chromatographically separated. Matrix selectivity studies showed endogenous chromatographic peak area ratios (PAR) at the analyte retention times were<20% of the analyte limit of quantitation PAR. The intra-assay accuracy ranged from 83.5% to 118% of target and the intra-run imprecision ranged from 2.0% to 19.1%. The inter-assay accuracy ranged from 90.3% to 104% of target and the inter-run imprecision ranged from 6.5% to 12.0%. Stability was established for 25 hours at room temperature, 207 days at -20°C, after three freeze-thaw cycles and for 26 days for rederivatized processed samples. After smoking marijuana predictable concentrations of THC, OH-THC and COOH-THC were seen; low concentrations of CBD were detected at early time points. In moderate users who had not smoked for at least 9 hours before ingesting an 800 mg oral dose of CBD, the method was sensitive enough to follow residual concentrations of THC and OH-THC; sustained COOH-THC concentrations over 50 ng/mL validated its higher analytical range.

Quantification of 11 Therapeutic Kinase Inhibitors in Human Plasma for Therapeutic Drug Monitoring Using Liquid Chromatography Coupled With Tandem Mass Spectrometry.

A liquid chromatography/tandem mass spectrometry assay was developed to facilitate therapeutic drug monitoring (TDM) for 10 anticancer compounds (dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, sorafenib, sunitinib, and vemurafenib) and the active metabolite, N-desethyl-sunitinib. The TDM assay is based on reversed-phase chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for analyte quantification. Stable isotopically labeled compounds were used as internal standards. The sample pretreatment consisted of protein precipitation with acetonitrile using a small plasma volume of 50 μL. The validation procedures were based on the guidelines on bioanalytical methods issued by the US Food and Drug Administration and were modified to fit the requirements of the clinical TDM environment. The method was validated over a linear range of 5.00-100 ng/mL for dasatinib, sunitinib, and N-desethyl-sunitinib; 50.0-1000 ng/mL for gefitinib and lapatinib; 125-2500 ng/mL for erlotinib, imatinib, and nilotinib; and 500-10,000 ng/mL for pazopanib, sorafenib, and vemurafenib. The results of the validation study demonstrated good intra-assay and interassay accuracy (bias <6.0%) and precision (12.2%) for all analytes. This newly validated method met the criteria for TDM and has successfully been applied to routine TDM service for tyrosine kinase inhibitors.

Detecting multiple lysosomal enzymes in dried blood spots by tandem mass spectrometry

Objective Establish a method for measuring the activities of Galactocerebrosidase (GALC), α-Glucosidase(GAA), α-Galactosidase (GLA) and α-L-Iduronidase (IDUA) in dried blood spots specimen by tandem mass spectrometry (MS/MS). Methods A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013.And twenty dried blood spot samples from patients withlysosomalstorage disorders(LSDs)of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014. The extraction of DBS was incubated with enzyme substrates and internal standards.After liquid-liquid and solid-phase extraction, the extraction solution was dried under nitrogen and reconstituted. Then enzyme reaction products and internal standards were analyzed by MS/MS. Linearity, precision, accuracy and the limit of detection were evaluated. 2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99.5th percentiles. 20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment. Results The intraassay and interassay precisions ranged from 1.7% to 11.8%, and the intraassay and interassay accuracies ranged from 85% to 115%. The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0.997-0.999. The limits of detection forGALC, GAA, GLA and GLA were 0.03 μmol/(L·h), 0.09 μmol/(L·h), 0.12 μmol/(L·h) and 0.16 μmol/(L·h). The normal reference values for GALC, GAA, GLA and GLAwere 0.51-8.51 μmol(L·h), 1.99-22.22 μmol/(L·h), 1.68-41.59 μmol/(L·h) and 2.36-19.21 μmol/(L·h). The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range. The Krabbe, Pompe, Fabry, MPSⅠ patients can be effectively detected by this MS/MS method. Conclusions A MS/MS method for measuring GALC, GAA, GLA and IDUA enzyme activities in DBShas been established.(Chin J Lab Med, 2016, 39: 761-765) Key words: Lysosomal storage diseases; Tandem mass spectrometry; Dried blood spot testing; Galactosylceramidase; Alpha-Glucosidases; Alpha-Galactosidase; Iduronidase

Quantification of Tamoxifen Polymeric Nanoparticles in female rodent breast tissue by UPLC/ESI-Q-TOF MS/MS

Objective: The present work aims to develop and validate stability indicating a novel liquid chromatography method with applicability i.e. to study the pharmacokinetics factor as well as estimate Tamoxifen (TMX) in bio-analytes, using Ultra High Pressure Liquid Chromatography/ Mass Spectrometry (UPLC-ESI-Q-TOF-MS/MS) in plasma. Methods: A bioanalytical method based on UPLC-ESI-Q-TOF-MS/MS has been developed and validated for the quantitative determination of TMX in female mice plasma using Clomiphine as an Internal Standard (IS). After Liquid-liquid extraction (LLE), analyte and IS, chromatographic separation was achieved on ACQUITY UPLC BEH C18 Waters column with dimensions; 100 mm × 2.1 mm; 1.7 μm, isocratic mobile phase (Acetonitrile:2 mM ammonium formate: 90:10; v/v), and a flow rate of 0.25 mL min -1 . Results: The transitions occurred at m/z 372.1→178.1 and m/z 407.1 → 100.1 for TMX and IS, respectively. Liquidliquid extraction technique (LLE) using ethyl acetate was applied in order to optimize the recovery of analytes in mice plasma. The run and retention time of TMX were 6.0 and 2.63 minutes, respectively while the linear dynamic range established was 1.002-4001.07 ng/ml (r2>0.998 ± 0.0003). Intra-assay and inter-assay accuracy (% RSD) was found in the range; 2.43- 3.49. Conclusion: The proposed LC–MS/MS assay method is simple, rapid and sensitive for the determination of TMX in mice plasma for pharmacokinetic studies. Analytes were stable under various conditions i.e. autosampler, freeze–thaw, at room temperature, and under deep freeze conditions. Key words Tamoxifen, Breast Cancer, UPLC/ESI-Q-TOF-MS/MS method Validation, Polymeric nanoparticles, Pharmacokinetics.

A GC-MS Method for Detection and Quantification of Cathine, Cathinone, Methcathinone and Ephedrine in Oral Fluid.

The stimulating herbal drug khat (cathine, cathinone) and its analog methcathinone are common substances of abuse in most countries. A GC-MS method was developed and validated for the detection and quantification of cathine, cathinone, methcathinone and ephedrine in oral fluid specimens. The analytes and internal standard (amphetamine-d5) were extracted from 0.5 mL oral fluids by ethyl acetate, and then the dried extracts were derivatized with heptafluorobutyric anhydride at 70°C for 30 min. The MS was used in selected ion monitoring mode. Ions monitored were m/z 117, 240 and 330 for cathine, m/z 77, 105 and 240 for cathinone, m/z 105, 210 and 254 for methcathinone, m/z 210, 254 and 344 for ephedrine and m/z 244 and 336 for amphetamine-d5 The calibration curves were linear (r(2)> 0.98) in the concentration range 20-2,000 ng/mL for all analytes. The intra- and inter-assay imprecisions were within (1.6-12.5%) and (1.5-9.5%), respectively, for all analytes. Intra-assay accuracies were between -5.9 and 6.7% for all analytes. The method was successfully applied to detect and quantify the target analytes from oral fluid specimens collected from Khat and methcathinone users.

A rapid and sensitive LC-MS/MS-ESI method for the determination of tolvaptan and its two main metabolites in human plasma.

A liquid chromatography-tandem mass spectrometry (LC-MS) method to quantify tolvaptan and its two main metabolites and applied to human study was first developed and validated as a measure of compliance in clinical research. Because of the structure similarity of tolvaptan and its multiple metabolites, the method was optimized to obtain a chromatographic and MS separation of the endogenous interference and isotope ions as well as high analysis throughput. Tolvaptan, its two main metabolites and the internal standard were extracted from human serum (0.1mL) using solid-phase extraction, separated on a Waters nova-pak C18 column (150×3.9mm, 5μm) using isocratic elution with a mobile phase composed of acetonitrile, water and formic acid (65:35:0.25, v/v/v). The total run-time was shortened to 3.5min. The mass transition ranges under positive electrospray ionisation that were monitored for quantitation included m/z 449-252 for tolvaptan, m/z 479-252 for metabolite DM-4103, m/z 481-252 for metabolite DM-4107 and m/z 463-266 for the internal standard (IS). The limit of quantification in plasma for all three analytes was 1ng/mL. The method was validated over a linear range from 1 to 500ng/mL for all three analytes with acceptable inter- and intra-assay precision and accuracy. The stability of the analytes was determined to be suitable for routine laboratory practices. The method was successfully applied to samples taken from research volunteers who ingested a 15mg tolvaptan tablet.

Simultaneous measurement of etravirine, maraviroc and raltegravir in pigtail macaque plasma, vaginal secretions and vaginal tissue using a LC-MS/MS assay.

Etravirine (ETR), maraviroc (MVC) and raltegravir (RAL) are promising antiretroviral drugs being used in HIV treatment and may be interesting for prevention applications such as oral or topical pre-exposure prophylaxis. Here we describe a sensitive and accurate method for the simultaneous detection of ETR, MVC and RAL from pigtail macaque plasma, vaginal secretions, and vaginal tissue. This method is characterized by a straightforward precipitation extraction method, a limit of quantification <0.5ngmL(-1) for all three antiretrovirals bolstered by a corresponding internal standard for each drug analyte, and short run time. Quantification is performed using positive ion electrospray triple quadrupole mass spectrometry. This method was validated over clinically relevant ranges for the three ARV drugs in all three matrices: 0.1-100ngmL(-1) for ETR, 0.05-100ngmL(-1) for MVC and 1-100ngmL(-1) for RAL. Our method is accurate and precise, with measured mean inter-assay precision (%CV) and accuracy (% bias) of 5.08% and 1.96%, respectively, while the mean intra-assay precision and accuracy were 3.44% and 1.08%. The overall post-extraction recovery for ETR, MVC and RAL was >94% in all cases. We also show that extracted biological samples are stable after storage at room temperature or 4°C and after three freeze/thaw cycles. This is the first analytical method capable of quantifying ETR, MVC and RAL in biological matrices relevant for pre-clinical testing of oral or topical HIV prevention methods in pigtailed macaques.

Plasma eicosanoid profiles determined by high-performance liquid chromatography coupled with tandem mass spectrometry in stimulated peripheral blood from healthy individuals and sickle cell anemia patients in treatment.

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Plasma interleukin-6 concentration in Standardbred racehorses determined by means of a novel validated ELISA.

To evaluate plasma interleukin 6 (IL-6) concentration in Standardbred racehorses by means of a novel ELISA following validation of the assay for use with equine plasma samples. Plasma samples obtained from 25 Thoroughbreds for use in assay validation and from 319 Standardbred racehorses at rest 2 to 2.5 hours prior to warm-up and racing. A sandwich ELISA was developed with equine anti-IL-6 polyclonal antibody and the biotin-streptavidin chemical interaction to enhance sensitivity. The assay was validated for specificity, sensitivity, precision, and accuracy by use of both recombinant and endogenous proteins. For the assay, cross-reactivity with other human and equine cytokines was very low or absent. Serial dilution of plasma samples resulted in proportional decreases in reactivity, indicating high specificity of the method. Partial replacement of detection antibody with capture antibody or pretreatment of samples with capture antibody caused assay signals to significantly decrease by 55%. The inter- and intra-assay precisions were ≤ 13.6% and ≤ 9.3%, respectively; inter- and intra-assay accuracies were within ranges of ± 14.1% and ± 8.6%, respectively, at concentrations from 78 to 5,000 pg/mL, and the sensitivity was 18 pg/mL. Plasma IL-6 concentration varied widely among the 319 Standardbreds at rest (range, 0 to 193,630 pg/mL; mean, 6,153 pg/mL; median, 376 pg/mL). This ELISA method proved suitable for quantification of IL-6 concentration in equine plasma samples. Plasma IL-6 concentration was high (> 10,000 pg/mL) in 9.1% of the Standardbred racehorses, which warrants further investigation.

Application of a validated LC–MS/MS method for JWH-073 and its metabolites in blood and urine in real forensic cases

Synthetic cannabinoids, which were synthesized to improve the therapeutic effects of cannabis, have become a major issue when they are abused. They have different chemical structures from tetrahydrocannabinol (THC) but similar effects on endocannabinoid receptors. “Spice” named products have more serious side effects than cannabis and can even cause death. These mixtures are prepared by spraying chemicals onto small pieces of herbs and are being dishonestly sold as “natural” and “legal” products over the internet. Their popularity is continuously increasing. Studies on detecting synthetic cannabinoids in biological samples as well as pharmacology and toxicology studies of these chemicals are very limited.A fast, specific and robust method for the detection and quantification of JWH-073, JWH-073 N-butanoic acid, and JWH-073 N-(4-hydroxybutyl) in blood and urine has been developed that uses solid-phase extraction (SPE) followed by UPLC–MS/MS analysis. This method has been validated in terms of its linearity (0.1–50ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<10%), recovery (75-95%), limits of detection (LODs) (0.08-0.13ng/mL), and limits of quantification (LOQs) (0.11-0.17ng/mL). Matrix effects, stability, and process efficiency parameters of this method have also been assessed. This method was applied to 2596 authentic samples received by the Department of Toxicology (Istanbul) in the Presidency of Council of Forensic Medicine (Turkey) between September 1, 2012, and February 28, 2015.

Monitoring IgA multiple myeloma: immunoglobulin heavy/light chain assays.

The use of electrophoresis to monitor monoclonal immunoglobulins migrating in the β fraction may be difficult because of their comigration with transferrin and complement proteins. Immunoassays specific for IgGκ, IgGλ, IgAκ, IgAλ, IgMκ, and IgMλ heavy/light chain (HLC) were validated for use in the clinical laboratory. We assessed sample stability, inter- and intraassay variability, linearity, accuracy, and reference intervals for all 6 assays. We tested accuracy by verifying that the sum of the concentrations for the HLC-pairs accounted for the total immunoglobulins in each of 129 healthy sera, and that the HLC-pair ratios (rHLCs) were outside the reference interval in 97% of 518 diagnostic multiple myeloma (MM) samples. We assessed diagnostic samples and posttreatment sera in 32 IgG and 30 IgA patients for HLC concentrations, rHLC, and total immunoglobulins and compared these nephelometry results with serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). In sample sets from patients with IgG MM, the sensitivity of SPEP was almost the same as for rHLC, and no additional advantage was conferred by running HLC assays. In pre- and posttreatment samples from patients with IgA MM, the SPEP, rHLC, and IFE identified clonality in 28%, 56%, and 61%, respectively. In addition, when M-spikes were quantifiable, the concentration of the involved HLC was linearly related to that of the SPEP M-spike, with a slope near 1. The use of IgA HLC assays for monitoring β-migrating IgA monoclonal proteins can substitute for the combination of SPEP, IFE, and total IgA quantification.

Validation of JWH-018 and its metabolites in blood and urine by UPLC–MS/MS: Monitoring in forensic cases

The herbal products referred to as ‘Spice’ have been used as ‘legal alternatives’ to cannabis worldwide since 2004. The first synthetic cannabinoid JWH-018 was detected in ‘Spice’ products in 2008, and has been banned by many legal authorities since the beginning of 2009. In order to prove use of JWH cannabinoids (JWHs), specific and robust methods were needed. We have developed a specific and reliable method for the detection and quantification of JWH-018, JWH-018 N-pentanoic acid, and JWH-018 N-(5-hydroxypentyl) in blood and urine using solid-phase extraction followed by UPLC–MS/MS analysis. The method has been validated in terms of linearity (0.1–50ng/mL), selectivity, intra-assay and inter-assay accuracy and precision (CV<15%), recovery (85–98%), limits of detection (LOD) (0.08–0.14ng/mL), and quantification (LOQ) (0.10–0.21ng/mL). Matrix effects, stability, and process efficiency were also assessed. The method has been applied to 868 authentic samples received by the Department of Chemistry (Istanbul) in the Council of Forensic Medicine of the Ministry of Justice.

LC–MS/MS method for the determination of two metabolites of tribendimidine, deacylated amidantel and its acetylated metabolite in plasma, blood and dried blood spots

Tribendimidine has emerged as potential alternative to praziquantel in the treatment of Opisthorchis viverrini infections. To support its clinical development program, a quantitative high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay was developed for tribendimidine's degradation product deacylated amidantel (dADT) and its acetylated metabolite adADT. Analytical sample preparation included protein precipitation for blood and plasma, and direct processing of dried blood spots (DBS). The analytes were detected by multiple reaction monitoring with electrospray ionization in the positive mode (dADT: 178.3→133.1m/z, adADT: 220.4→175.1m/z, tribendimidine 294.3→249.0m/z). A pentafluorophenyl (PFP) phase Kinetex analytical column (2.6μm, 100Å, 50mm×4.6mm) with a 6min lasting mobile phase gradient program of ammonium acetate and acetonitrile was applied. The method was validated with respect to precision, accuracy, linearity, sensitivity, and selectivity. The analytical range in plasma and blood was 1–1000ng/ml and in DBS 10–2000ng/ml (R2>0.99). Recoveries determined using four different human blood batches were in the range of 70–90%. Inter- and intra-assay accuracy and precision deviations were at least ≤12.2%. dADT and adADT were stable within the autosampler for 72h (10°C), for 4h at room temperature, for 3 month at −80°C, and after three freeze and thaw cycles. DBS samples should be stored at −20°C. The validation results demonstrated that the LC–MS/MS method is precise, accurate and selective and can be applied for pharmacokinetic studies with tribendimidine.

Evaluation of the Analytical Performance of the Modified Enterprise Point-of-Care Blood Gas and Electrolyte Analyzer in a Pediatric Hospital

Arterial blood gas testing is an essential component that directs the evaluation of a patient’s general condition, especially in the emergent setting. We evaluated the analytical performance of the modified Enterprise point-of-care (EPOC) blood gas and electrolyte analyzer system with respect to precision and accuracy and compared our results with those obtained using the point-of-care i-STAT method and the criterion standard ABL 835 Radiometer blood gas analyzer in a pediatric hospital. Evaluation studies were performed as per the Clinical Laboratory Standards Institute guidelines and included intra-assay and interassay precision and accuracy, linearity, and concordance analyses with i-STAT and ABL 835 Radiometer. Intra-assay and interassay coefficient of variation for each analyte was calculated and was less than 6% for all analytes in the ranges tested including pH, Po2, Pco2, Na+, K+, Ca2+, glucose, lactate, and hematocrit, Cl−, and creatinine. All of the analytes were linear across the reportable range. For the methods comparison of EPOC system with the i-STAT method, using 40 samples, the correlation coefficients were r > 0.94 for all analytes with the exception of Ca2+ (r = 0.61). For the methods comparison of EPOC system with the criterion standard ABL 835 Radiometer on 40 samples, the correlation coefficients were higher than 0.97 for all analytes with the exception of hematocrit and hemoglobin (r = 0.8). Thus, in our pediatric hospital setting, the EPOC system showed excellent precision and accuracy and compared favorably with the i-STAT and ABL 835 Radiometer assay results. These findings, together with the low cost, room temperature storage of test cards and availability of metabolites such as lactate, glucose, and creatinine along with the wireless connectivity of the EPOC system, provide an operational advantage over other point-of-care blood gas analyzers currently available.

Quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma by liquid chromatography/tandem mass spectrometry

Therapeutic drug monitoring is a cornerstone of antibacterial therapy, especially in an era of increasing antibacterial resistance in individualizing antimicrobial therapy. Liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay was used for the simultaneous measurement of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in 50 μl plasma samples over a wide range. The overall turnaround time for the assay was 20 minutes. Intra-assay precision and accuracy for quality control samples ranged within 1·8–8·5 and 91·4–106·7%, respectively. Inter-assay precision and accuracy ranged within 1·3–14·4 and 95·8–104·6%, respectively. The lower limit of quantification was below 1·5 μg/ml for all the five antibiotics. No ion suppression due to matrix effects was found. A simple and rapid LC–MS/MS method which provides high specificity, precision and accuracy for the simultaneous quantification of piperacillin, tazobactam, meropenem, ceftazidime, and linezolid in human plasma has been developed and validated. The present method is suitable for therapeutic drug monitoring in paediatrics.