Initial experience regarding the safety and yield of rest-stress myocardial perfusion imaging in emergency department patients with mildly abnormal high-sensitivity cardiac troponins.

BACKGROUND: With high-sensitivity troponin testing, approximately a third of patients presenting to emergency departments (EDs) with suspected acute coronary syndromes will have mildly abnormal values. However, data regarding rest-stress myocardial perfusion imaging (MPI) in these patients are limited. We hypothesize that stress testing is safe and that the yield for detecting myocardial ischemia is associated with risk stratification by the HEART score. METHODS AND RESULTS: We conducted a retrospective cohort study of consecutive patients referred for rest-stress MPI with mildly abnormal high-sensitivity troponin T (hs-cTn) values. Outcomes were adverse events related to stress MPI, defined as myocardial infarction or ventricular tachyarrhythmia, and the presence of ischemia, defined as a reversible perfusion defect. Among 213 patients, the median age was 67, most were male (61.5%, n = 131), and prior CAD was common (53.5%, n = 114). Myocardial ischemia was present in 13.6% (n = 29), and there were no adverse events attributable to stress MPI. A higher HEART score was associated with myocardial ischemia (Odds Ratio [OR] 1.50, 95% Confidence Interval [CI] 1.08 to 2.08, P = .002). CONCLUSION: Rest-stress MPI appears safe in patients with mildly abnormal hs-cTn values, and the yield for detecting ischemia is associated with the HEART score, though further validation studies are needed.

A robust LC-MS/MS assay with online cleanup for measurement of serum testosterone.

Accurate measurement of low levels of testosterone is critical for diagnosis and treatment of androgen disorders. The very low concentrations of testosterone in children, females, and males with androgen suppression therapies necessitate the use of mass spectrometry-based methods. We aimed to develop a liquid chromatography with tandem mass spectrometry method with simplified sample preparation and online solid-phase extraction cleanup to achieve enhanced precision, accuracy, robustness, and cost-effectiveness. The assay was linear from 10 to 20 000 pg/mL with an analytical recovery of 93-104%. The total coefficient of variation was 2.5, 1.9, and 1.7% at concentration levels of 348, 5432, and 10 848 pg/mL, respectively. No significant carryover was observed from samples with concentrations up to 20 000 pg/mL. No significant interference was observed from androstenedione, dehydroepiandrosterone, epi-testosterone, and estriol. Comparison with CDC Hormone Standardization program (HoSt) reference samples with defined values (n = 40) showed a Deming regression slope of 0.963, intercept of 28.06 pg/mL, standard error of estimate was 66.9, a correlation coefficient of 0.9996, and a mean bias of -0.6%. The method met the accuracy criteria by the CDC HoSt program. In addition, we achieved >12 000 injections on a single analytical column without significant performance deterioration due to the specific online solid-phase extraction settings.

Multicenter evaluation of the thermo scientific prelude for measurement of immunosuppressant drugs using sample preparation liquid chromatography-tandem mass spectrometry.

Immunosuppressant drugs (ISDs) are commonly prescribed to solid organ transplant patients. Their narrow therapeutic index and potential for toxicity necessitates careful monitoring of blood concentrations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are increasingly used for ISD measurement. However, there remain many challenges with this methodology, particularly regarding interassay variability. The Thermo Scientific Prelude is an online extraction/liquid chromatography platform that uses turbulent flow technology coupled with MS/MS. A multicenter evaluation of the Prelude for the measurement of cyclosporine A, tacrolimus, and sirolimus is described. ISDs were measured at each site using standardized protocols. Sample preparation liquid chromatography-MS/MS was performed using the Prelude coupled to a TSQ Vantage. Chromatography was achieved with a Cyclone-P TurboFlow/Accucore C8 column combination using a multisolvent loading and eluting pump system. Mass spectrometry acquisitions were performed in selective reaction monitoring mode and data processed using TraceFinder (version 3.1). Multisite mean imprecision for cyclosporine A ranged from 8.8% (54 mcg/L) to 9.8% (450 mcg/L); for tacrolimus, 4.7% (15.5 mcg/L) to 12.6% (2.5 mcg/L); for sirolimus, 7.4% (19.9 mcg/L) to 16.5% (2.6 mcg/L). Approximately 110 specimens were used for method comparison. For cyclosporine A, mean bias against the multisite mean ranged from -18% to 1%; for tacrolimus, values ranged from -7% to 4%; for sirolimus, values ranged from -4% to 2%. Comparisons of multisite mean Prelude results with routine ISD method results was also performed for cyclosporine A (slope = 0.7878, intercept = 24.16, r = 0.98), tacrolimus (slope = 0.9391, intercept = 0.1017, r = 98), and sirolimus (slope = 0.9618, intercept = 0.1483, r = 0.97). The Prelude ISD method offers acceptable and comparable multisite performance. This study has also highlighted the importance of adopting standardized protocols and LC-MS/MS methods for better comparability between ISD assays.

Applicability of chronic kidney disease epidemiology collaboration equations in a Chinese population.

BACKGROUND: Accurate estimated glomerular filtration rates (eGFR) is an important step in the diagnosis of chronic kidney disease (CKD). The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, based on creatinine alone (eGFRcr), was developed to improve on the Modification of Diet in Renal Disease equation, in particular by addressing the systematic underestimation of high GFR. Whether the CKD-EPI equation, based on cystatin C alone (eGFRcys), or the combined creatinine-cystatin C CKD-EPI equation (eGFRcr-cys C), actually perform better than the CKD-EPI equation based on creatinine (eGFRcr) remains unknown, especially in Asians including Chinese populations, where eGFR equations may overestimate true GFR. METHODS: A standard dual plasma sampling method (DPSM) of estimating (99m)Tc-diethylene triamine penta-acetic acid clearance was used to determine the reference or measured GFR (mGFR). Linear regression analysis, Bland-Altman analysis, bias, absolute bias and accuracy (P30) were used to compare the performance of the combined creatinine-cystatin C equation (eGFRcr-cys) and equations based on each marker alone (eGFRcr and eGFRcys) in Chinese subjects, including both patients with CKD and healthy individuals. RESULTS: We enrolled 617 Chinese participants (49.11% female, 47.11 ± 17.25 years old), with a mean mGFR of 73.80 ± 37.55 mL/min/1.73 m(2). The predictive abilities (r), the accuracy (P15, P30, P50), bias and absolute bias of the eGFRcr-cys equation were superior to eGFRcr equation and the eGFRcys equation in overall samples. Bland-Altman analysis also demonstrated a consistent result. When compared in subgroups, the accuracy (P30) of all three equations exceeded 90% at mGFR ≥90 mL/min/1.73m(2); the eGFRcr-cys equation had the highest accuracy (P30: 95.56%). At mGFR 60-89 mL/min/1.73 m(2), the accuracies (P30) of the eGFRcr-cys and eGFRcr equations exceeded the acceptable level (≥70%), and there was no significant difference between them (P = 0.58). At mGFR <60 mL/min/1.73 m(2), the accuracy (P30) of all three equations was below 70%, but the eGFRcr-cys equation had the greatest precision. CONCLUSIONS: The performances of the eGFRcr-cys and eGFRcr equations were similar to superior to that of the eGFRcys equation at higher GFR levels in an Asian population, especially in normal and mild to moderate kidney disease. Further improvement is needed for these equations at GFR <60 mL/min per 1.73 m(2).

Fast, simple, and sensitive high-performance liquid chromatography method for measuring vitamins A and E in human blood plasma.

Vitamins A and E are fat-soluble vitamins that play important roles in several physiological processes. Monitoring their concentrations is needed to detect deficiency and guide therapy. In this study, we developed a high-performance liquid chromatography method to measure the major forms of vitamin A (retinol) and vitamin E (α-tocopherol and γ-tocopherol) in human blood plasma. Vitamins A and E were extracted with hexane and separated on a reversed-phase column using methanol as the mobile phase. Retinol was detected by ultraviolet absorption, whereas tocopherols were detected by fluorescence emission. The chromatographic cycle time was 4.0 min per sample. The analytical measurement range was 0.03-5.14, 0.32-36.02, and 0.10-9.99 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. Intr-aassay and total coefficient of variation were <6.0% for all compounds. This method was traceable to standard reference materials offered by the National Institute of Standards and Technology. Reference intervals were established using plasma samples collected from 51 healthy adult donors and were found to be 0.30-1.20, 6.0-23.0, and 0.3-3.2 mg/L for retinol, α-tocopherol, and γ-tocopherol, respectively. In conclusion, we developed and validated a fast, simple, and sensitive high-performance liquid chromatography method for measuring the major forms of vitamins A and E in human plasma.

Comparison of a stable isotope-labeled and an analog internal standard for the quantification of everolimus by a liquid chromatography-tandem mass spectrometry method.

BACKGROUND: Everolimus is an immunosuppressant drug used in solid organ transplantation. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) methods have been used for therapeutic drug monitoring of this drug. In LC-tandem mass spectrometry (MS/MS) methods, both 32-desmethoxyrapamycin and everolimus-d4 have been used as internal standards. OBJECTIVES: To compare 2 internal standards (32-desmethoxyrapamycin and everolimus-d4) for the quantification of everolimus by an LC-MS/MS method. METHODS: Both 32-desmethoxyrapamycin and everolimus-d4 were introduced in the method validation process with 2 transitions simultaneously monitored for everolimus (975.6 → 908.7 as the quantifier and 975.6 → 926.9 as the qualifier) by an established LC-MS/MS method. The key performance characteristics were lower limit of quantification, accuracy, precision, and comparison with an LC-MS/MS method offered by another laboratory. RESULTS: The lower limit of quantification (LLOQ) was 1.0 ng/mL using either internal standard with an analytical recovery of 98.3%-108.1% across the linear range. The total coefficient of variation for everolimus was 4.3%-7.2% with no significant difference between the 2 internal standards. In comparison with an independent LC-MS/MS method, though everolimus-d4 offered a better slope (0.95 versus 0.83), both internal standards showed acceptable results and had a coefficient of correlation r > 0.98 in the tested concentration range of 1.2-12.7 ng/mL. CONCLUSIONS: Although everolimus-d4 offered a more favorable comparison with an independent LC-MS/MS method, both everolimus-d4 and 32-desmethoxyrapamycin had acceptable performance as the internal standards for everolimus quantification by the LC-MS/MS method.

Applications of monolithic solid-phase extraction in chromatography-based clinical chemistry assays.

Complex matrices, for example urine, serum, plasma, and whole blood, which are common in clinical chemistry testing, contain many non-analyte compounds that can interfere with either detection or in-source ionization in chromatography-based assays. To overcome this problem, analytes are extracted by protein precipitation, solid-phase extraction (SPE), and liquid-liquid extraction. With correct chemistry and well controlled material SPE may furnish clean specimens with consistent performance. Traditionally, SPE has been performed with particle-based adsorbents, but monolithic SPE is attracting increasing interest of clinical laboratories. Monoliths, solid pieces of stationary phase, have bimodal structures consisting of macropores, which enable passage of solvent, and mesopores, in which analytes are separated. This structure results in low back-pressure with separation capabilities similar to those of particle-based adsorbents. Monoliths also enable increased sample throughput, reduced solvent use, varied support formats, and/or automation. However, many of these monoliths are not commercially available. In this review, application of monoliths to purification of samples from humans before chromatography-based assays will be critically reviewed.

A simple, fast, and sensitive method for the measurement of serum nicotine, cotinine, and nornicotine by LC-MS/MS.

The measurement of nicotine and its metabolites has been used to monitor tobacco use. A high-sensitivity method (<1 ng/mL) is necessary for the measurement in serum or plasma to differentiate nonsmokers from passive smokers. Here, we report a novel LC-MS/MS method to quantify nicotine, cotinine, and nornicotine in serum with high sensitivity. Sample preparation involved only protein precipitation, followed by online turbulent flow extraction and analysis on a porous graphitic carbon column in alkaline conditions. The chromatography time was 4 min. No significant matrix effects or interference were observed. The lower limit of quantification was 0.36, 0.32, and 0.38 ng/mL for nicotine, cotinine, and nornicotine, respectively, while accuracy was 91.6-117.1%. No carryover was observed up to a concentration of 48 , 550, and 48 ng/mL for nicotine, cotinine, and nornicotine, respectively. Total CV was <6.5%. The measurement of nicotine and cotinine was compared with an independent LC-MS/MS method and concordant results were obtained. In conclusion, this new method was simple, fast, sensitive, and accurate. It was validated to measure nicotine, cotinine, and nornicotine in serum for monitoring tobacco use.

A systematic examination of anti-drug antibody titer estimation: Applied recommendations.

Biologic drugs (therapeutic proteins or peptides) have become one of the most important therapeutic modalities over the past few decades. Drug-induced immunogenicity is a significant concern as it may affect safety, tolerability, and efficacy. With more sensitive and drug-tolerant screening assays in use today, reliable estimation of anti-drug-antibody (ADA) titer has become more important for understanding clinically relevant ADA levels. Titer is commonly defined as the dilution factor resulting in an assay signal equal to a pre-specified cut point factor. Given its influence on the resulting titer precision, the choice of a titer cut point factor warrants careful consideration. In this paper, we discuss the theoretical dilution model, investigate how titer variability depends on the cut point factor and propose a standardized cut point factor to increase precision of titer estimates. Additionally, we demonstrate that non-linear regression-based titer estimation provides both improved precision and implementation efficiency relative to commonly used estimation approaches.

Highly sensitive and selective measurement of underivatized methylmalonic acid in serum and plasma by liquid chromatography-tandem mass spectrometry.

Methylmalonic acid (MMA) is a functional biomarker of vitamin B12 deficiency. Measurement of plasma MMA is challenging due to its small molecular weight and hydrophilic nature. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for measuring plasma MMA. However, these methods involve lengthy sample preparation, long chromatographic run time, inadequate sensitivity, or interference from succinic acid (SA). Here we report a novel LC-MS/MS method for quantitation of underivatized MMA in serum or heparinized plasma with high sensitivity and selectivity. Sample preparation involved only strong anion exchange solid phase extraction. The extract was purified by online turbulent flow and analyzed on an Organic Acids column. MS/MS analysis was performed in negative electrospray mode, and the analytical time was 6 min. The use of ion ratio confirmation in combination with chromatographic resolution from SA greatly enhanced the selectivity. No interference was observed. This method was linear from 26.2 to 26,010.0 nM with an accuracy of 98-111 %. Total coefficient of variation was less than 4.6 % for three concentration levels tested. Comparison with a reference laboratory LC-MS/MS method using leftover patient serum specimens (n = 48) showed a mean bias of -2.3 nM (-0.61 %) with a Deming regression slope of 1.016, intercept of -6.6 nM, standard error of estimate of 25.3 nM, and a correlation coefficient of 0.9945. In conclusion, this LC-MS/MS method offers highly sensitive and selective quantitation of MMA in serum and plasma with simple sample preparation.

A simple and fast liquid chromatography-tandem mass spectrometry method for measurement of underivatized L-arginine, symmetric dimethylarginine, and asymmetric dimethylarginine and establishment of the reference ranges.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase and an established biomarker for endothelial function, while symmetric dimethylarginine (SDMA), an emerging biomarker for renal function, has been shown to outperform creatinine-based equations for estimated glomerular filtration rate. In order to study these analytes for clinical research, a fast and simple method for measuring arginine (ARG), SDMA, and ADMA in plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Plasma (50 µL) was mixed with 50 µL of internal standard of (13)C-arginine and d(7)-ADMA followed by protein precipitation with methanol containing 1% ammonium acetate (300 µL). After centrifugation, the supernatant (100 µL) was mixed with 300 µL of acetonitrile with 1% formic acid, and the mixture was injected onto a silica column monitored by a mass spectrometer. The analytical cycle time was 5.0 min. The method was linear from 5.7 to 489.7 µM for ARG, 0.06 to 5.15 µM for SDMA, and from 0.34 to 5.65 µM for ADMA, with an accuracy of 99.0-120.0%. Total coefficients of variation for all analytes ranged from 2.7% to 7.7% for three concentration levels. The effects of hemolysis, lipemia, uremia, icterus, specimen tube types, storage at different temperature, and freeze/thaw were thoroughly investigated. Reference ranges were established using 51 well-defined reference subjects (12 men and 39 women, age 19-64 years): 53.1-129.7 µM for ARG, 0.32-0.65 µM for SDMA, and 0.36-0.67 µM for ADMA. In conclusion, the validated LC-MS/MS method described here offers a fast and reliable ARG, SDMA, and ADMA quantitation in plasma with minimum sample preparation.

AACC Guidance Document on the Use of Point-of-Care Testing in Fertility and Reproduction.

BACKGROUND: The AACC Academy revised the reproductive testing section of the Laboratory Medicine Practice Guidelines: Evidence-Based Practice for Point-of-Care Testing (POCT) published in 2007. METHODS: A panel of Academy members with expertise in POCT and laboratory medicine was formed to develop guidance for the use of POCT in reproductive health, specifically ovulation, pregnancy, premature rupture of membranes (PROM), and high-risk deliveries. The committee was supplemented with clinicians having Emergency Medicine and Obstetrics/Gynecology training. RESULTS: Key recommendations include the following. First, urine luteinizing hormone (LH) tests are accurate and reliable predictors of ovulation. Studies have shown that the use of ovulation predicting kits may improve the likelihood of conception among healthy fertile women seeking pregnancy. Urinary LH point-of-care testing demonstrates a comparable performance among other ovulation monitoring methods for timing intrauterine insemination and confirming sufficient ovulation induction before oocyte retrieval during in vitro fertilization. Second, pregnancy POCT should be considered in clinical situations where rapid diagnosis of pregnancy is needed for treatment decisions, and laboratory analysis cannot meet the required turnaround time. Third, PROM testing using commercial kits alone is not recommended without clinical signs of rupture of membranes, such as leakage of amniotic fluid from the cervical opening. Finally, fetal scalp lactate is used more than fetal scalp pH for fetal acidosis due to higher success rate and low volume of sample required. CONCLUSIONS: This revision of the AACC Academy POCT guidelines provides recommendations for best practice use of POCT in fertility and reproduction.

The MMACHC proteome: hallmarks of functional cobalamin deficiency in humans.

Cobalamin (Cbl, B(12)) is an essential micronutrient required to fulfill the enzymatic reactions of cytosolic methylcobalamin-dependent methionine synthase and mitochondrial adenosylcobalamin-dependent methylmalonyl-CoA mutase. Mutations in the MMACHC gene (cblC complementation group) disrupt processing of the upper-axial ligand of newly internalized cobalamins, leading to functional deficiency of the vitamin. Patients with cblC disease present with both hyperhomocysteinemia and methylmalonic acidemia, cognitive dysfunction, and megaloblastic anemia. In the present study we show that cultured skin fibroblasts from cblC patients export increased levels of both homocysteine and methylmalonic acid compared to control skin fibroblasts, and that they also have decreased levels of total intracellular folates. This is consistent with the clinical phenotype of functional cobalamin deficiency in vivo. The protein changes that accompany human functional Cbl deficiency are unknown. The proteome of control and cblC fibroblasts was quantitatively examined by two dimensional difference in-gel electrophoresis (2D-DIGE) and liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. Pathway analysis of the differentially expressed proteins demonstrated strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases in the cblC mutant cell lines. Supplementation of the cell cultures with hydroxocobalamin did not restore the cblC proteome to the patterns of expression observed in control cells. These results concur with the observed phenotype of patients with the cblC disorder and their sometimes poor response to treatment with hydroxocobalamin. Our findings could be valuable for designing alternative therapies to alleviate the clinical manifestation of the cblC disorder, as some of the protein changes detected in our study are common hallmarks of known pathologies such as Alzheimer's and Parkinson's diseases as well as muscular dystrophies.

Sensitive measurement of serum 1α,25-dihydroxyvitamin D by liquid chromatography/tandem mass spectrometry after removing interference with immunoaffinity extraction.

Vitamin D plays important roles in bone health and a variety of other pathophysiological conditions. 1α,25-Dihydroxyvitamin D is the active form of vitamin D. Quantification of serum 1α,25-dihydroxyvitamin D is useful for evaluation of several diseases including chronic renal failure, hypoparathyroidism, sarcoidosis, and rickets. Measurement of 1α,25-dihydroxyvitamin D is very challenging due to its low circulating concentration and presence of interfering substances in serum. In this report, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantifying serum 1α,25-dihydroxyvitamin D is described. Lithium adducts of 1α,25-dihydroxyvitamin D were formed prior to mass spectrometry analysis to improve ionization efficiency. We tested a number of different sample preparation procedures and found that immunoaffinity extraction was the method of choice because it completely removed isobaric interferences and matrix effects present in patient serum. Extraction efficiency, expressed as absolute recovery, was greater than 60% in both patient serum and charcoal-stripped serum. This method was linear from 3.4 to 206.2 pg/mL for 1α,25-dihydroxyvitamin D(3) and 3.9 to 212.6 pg/mL for 1α,25-dihydroxyvitamin D(2) with an accuracy of 89.8-98.4% and 97.5-115.7%, respectively. Inter-assay and intra-assay coefficients of variance (CVs) for both analytes at two different concentration levels ranged from 2.5-7.0%. Comparison with a radioimmunoassay for measuring total 1α,25-dihydroxyvitamin D concentration using 40 patient samples showed a Deming regression slope of 0.751, a y-intercept of 0.84 pg/mL, an r value of 0.7909, and a mean percentage difference of -27.1%. Comparison with a reference LC/MS/MS method (n = 20) showed a Deming regression slope of 1.020, a y-intercept of 1.32 pg/mL, an r value of 0.9797, and a mean percentage difference of -2.9%. In conclusion, usage of immunoaffinity extraction enabled a sensitive LC/MS/MS method for quantification of 1α,25-dihydroxyvitamin D in serum.

Performance characteristics of a new levetiracetam immunoassay and method comparison with a high-performance liquid chromatography method.

BACKGROUND: Although levetiracetam is recognized for ease of dosing and being well tolerated, therapeutic drug monitoring is potentially useful in certain clinical situations. High-performance liquid chromatography has been commonly used for measuring levetiracetam. Recently, a homogeneous immunoassay for levetiracetam measurement in serum and plasma was introduced by ARK Diagnostics, Inc. The goal of this work was to validate this assay on a random access instrument. DESIGN AND METHODS: This immunoassay was established on the Siemens ADVIA 1200 automated chemistry analyzer. The intraday precision was assessed by 10 replicates of two levels of quality control materials in a batch, whereas interday precision was estimated by assaying the same materials one set per day for 20 days. Linearity was evaluated by serially diluting the highest calibrator and a high patient specimen run in triplicate, whereas the lower limit of quantification was confirmed by 10 measurements of a low-level specimen diluted from a calibrator and another from a diluted patient specimen. This method was compared with a commercial high-performance liquid chromatography method (Chromsystems) using 63 specimens from patients who were on levetiracetam therapy. RESULTS: The assay cycle was 10 minutes with a theoretical throughput of 800 per hour. The intra- (n = 10) and interday (n = 20) coefficients of variation were 8.1% or less for the two levels tested. The manufacturer-claimed analytical measurable range (2.0-100.0 µg/mL) was confirmed by serial dilution and lower limit of quantification experiments. Among the 63 patient samples studies, four showed levetiracetam levels below 2.0 µg/mL by both methods. Deming regression using the remaining 59 paired patient results by ARK immunoassay and the high-performance liquid chromatography method showed a correlation coefficient of 0.9962, a linear regression slope of 0.98, and an intercept of 0.61 with a mean bias of 0.04%. CONCLUSION: The ARK immunoassay is suitable for clinical use of monitoring levetiracetam levels in serum/plasma on an automated chemistry analyzer (Siemens ADVIA 1200).

Simultaneous quantification of 19 drugs/metabolites in urine important for pain management by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Monitoring pain management drugs and frequently abused drugs is important for physicians to assess patient compliance. Liquid chromatography-tandem mass spectrometry offers high specificity needed for this purpose. In this report, a novel liquid chromatography-tandem mass spectrometry method for simultaneously monitoring 19 drugs/metabolites in urine was developed and validated. METHODS: Sample preparation included hydrolysis, dilution and turbulent flow online extraction. Analysis was achieved by reverse phase liquid chromatography and triple-quadruple tandem mass spectrometry. Two fragment ions, one for quantification and the other for assuring identification, were monitored for each analyte. RESULTS: No matrix effect or interference was observed. Lower limits of quantification ranged from 5 to 25 ng/mL. Within the linear range, analytical recovery was between 85.8% and 119.4%. Intra-assay and total coefficient of variations were between 0.2% and 12.7%. This method was compared with mass spectrometry methods offered by two other laboratories using 82 patient samples and 60 spiked urine samples showing 60%-100% agreement. The current method identified more positive samples for all analytes except THCA. The discrepancy in detection rates was primarily due to the different cut-offs used by the other laboratories. CONCLUSIONS: A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed for measuring 19 drugs/metabolites in urine important for pain management clinics.

A simple liquid chromatography-tandem mass spectrometry method for measuring metanephrine and normetanephrine in urine.

BACKGROUND: Measuring urinary fractionated metanephrines is one of the initial tests in the diagnosis of pheochromocytoma. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) represents the most specific and accurate technology for this purpose. The goal of this work was to develop a simple LC-MS/MS method for measuring metanephrines in urine. METHODS: Each urine sample was complexed with diphenylboronic acid, and purified on a Bond-Elute Plexa cartridge. The extract was concentrated and analyzed on a short Atlantis T3 column in 8.5 min. Metanephrines and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring. RESULTS: Ion suppression was observed, but was compensated for by the respective internal standard. The analytical measurement range was 0.2-27.4 µmol/L and 0.3-14.6 µmol/L for metanephrine and normetanephrine, respectively. The intra-assay and total coefficient of variation throughout the linear ranges was 2.03%-2.11% and 2.20%-3.80% for metanephrine, and 4.50%-8.09% and 9.00%-10.00% for normetanephrine, respectively. Comparison with a commercial HPLC method using patient samples (n=65) by Passing-Bablok regression showed a slope of 1.000 and 1.014, y-intercept of -0.080 and -0.067, a correlation coefficient of 0.8830 and 0.9022, and a mean difference of 14.0% and -0.43% for metanephrine and normetanephrine, respectively. CONCLUSIONS: This simple method for urine metanephrines is suitable for clinical use.

Applications of monolithic columns in liquid chromatography-based clinical chemistry assays.

Monolithic columns have slowly been applied to HPLC methods for clinical chemistry testing in the last 10 years. The application areas include therapeutic drug monitoring, drugs of abuse, vitamins, porphyrins, and steroids. In comparison with conventional particulate columns, the monolithic columns may offer shorter chromatography time, more robustness, and better resolution for certain analytes. The potential drawback of large mobile phase consumption may be improved with smaller id columns, which are currently on the market. Methods covered in this review are those searchable in PubMed up to December 2010. This review highlights the emergence of monolithic column technology in HPLC methods used for clinical chemistry testing. The goals of this review are threefold: (i) To identify the areas of clinical chemistry that analytical monolithic columns have been used in HPLC methods. (ii) To demonstrate the application of analytical monolithic columns in HPLC methods using different detection systems. (iii) To discuss the advantages and limitations of the monolithic columns compared with particulate columns in the clinical chemistry applications.

Real-World Clinical Performance Evaluation of a Fourth-Generation HIV Antigen/Antibody Differentiation Test.

BACKGROUND: HIV testing is still an important component of routine sexual health screening, assessment of at-risk individuals and as part of the care of pregnant women. To prevent further transmission of infection, it is important that HIV tests are highly sensitive and that positive cases are not missed. HIV serologic antigen/antibody tests are commonly used as they are capable of detecting recent and established infection. METHODS: In this study we assessed the performance of the Elecsys HIV Duo assay (Elecsys assay) against the Abbott Architect assay in 10 121 samples from US and non-US adult, pediatric, and pregnant populations including low-risk, high-risk, and known positive cohorts. Congruent repeatedly reactive and/or discrepant samples followed a confirmatory algorithm consisting of an antigen/antibody differentiation assay and a nucleic acid test, as per the study protocol. RESULTS: The overall sensitivity of the Elecsys assay was 100.00% (95% CI 99.81-100.00 [1977/1977]), and the specificity was 99.84% (95% CI 99.73-99.91 [8129/8142]). The Elecsys assay detected all positive samples within the study, including all 50 antigen-only positive samples and samples from different HIV subtypes, including group O, group M subtypes, HIV-2 positives, and HIV-1 and HIV-2 dual positives. CONCLUSIONS: The Elecsys HIV Duo assay was highly sensitive for diagnosis of HIV in a range of clinical samples from the United States and outside the United States and is suitable for routine use.

Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin-bound vitamin B(12).

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function.