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BACKGROUND Infants born preterm are affected by a hypothalamic-pituitary-thyroid axis that is immature and still developing as they progress closer to corrected term gestation. Multiple risk factors place preterm infants at risk for a hypothyroid state. However, there is variability in thyroid-stimulating hormone cutoff values and limited data on free thyroxine reference intervals to guide clinicians. METHODS 1584 thyroid-stimulating hormone and 1576 free thyroxine laboratory samples that were originally collected to screen hospitalized infants for delayed-onset of hypothyroidism were retrospectively evaluated from a group of 1087 infants who ranged in postmenstrual age from 25 to 43 weeks gestation at the time of laboratory sample collection. Median thyroid hormone values and reference intervals were established using R and the mixtools package. RESULTS Thyroid-stimulating hormone reference intervals remained similar across gestational ages from 0.340-9.681 µIU/mL in 25-27 6/7-week infants to 1.090-7.627 µIU/mL in 40-43-weeks infants. For the same age groups, free thyroxine reference intervals increased from 0.42-0.91 ng/dL to 0.87-1.32 ng/dL. CONCLUSION The reference intervals identified suggest that infants

Background Doctoral level board-certified clinical chemists play an invaluable role in many facets of laboratory medicine and healthcare. However, information concerning their total compensation is sparse. Content A confidential self-reported compensation survey was conducted by the American Association for Clinical Chemistry’s Society for Young Clinical Laboratorians (AACC SYCL) Core Committee from April 1 to April 17, 2018. Respondents provided information on geographic location, employment sector, gender, and years of experience to account for the influence of these variables on compensation. There were 199 respondents in total from the United States and Canada, however, only respondents employed in the United States with an earned doctoral degree and certification by the American Board of Clinical Chemistry (n = 133), were included in the full analysis. In comparison to compensation reported in AACC SYCL salary surveys conducted in 2010 and 2013, early career median salaries are trending upwards after correction for inflation. Summary This survey is the first to collect the gender of respondents, and identify a pay gap for some geographic groups. However, this gap could be due in part to a difference in the years of experience, since males were highly represented in the group with >20 years of experience (25 out of 35, 71%). Future studies on compensation trends within clinical chemistry that do not rely on self-report are needed to ensure accuracy and completeness of the dataset.

Infants born preterm are affected by a hypothalamic-pituitary-thyroid axis that is immature and still developing as they progress closer to corrected term gestation. Multiple risk factors place preterm infants at risk for a hypothyroid state. However, there is variability in thyroid-stimulating hormone cutoff values and limited data on free thyroxine reference intervals to guide clinicians.1584 thyroid-stimulating hormone and 1576 free thyroxine laboratory samples that were originally collected to screen hospitalized infants for delayed-onset of hypothyroidism were retrospectively evaluated from a group of 1087 infants who ranged in postmenstrual age from 25 to 43 weeks gestation at the time of laboratory sample collection. Median thyroid hormone values and reference intervals were established using R and the mixtools package.Thyroid-stimulating hormone reference intervals remained similar across gestational ages from 0.340-9.681 µIU/mL in 25-27 6/7-week infants to 1.090-7.627 µIU/mL in 40-43-weeks infants. For the same age groups, free thyroxine reference intervals increased from 0.42-0.91 ng/dL to 0.87-1.32 ng/dL.The reference intervals identified suggest that infants31 weeks gestation have a higher thyroid-stimulating hormone and lower free thyroxine level at baseline than previously anticipated.The increasing free thyroxine values in preterm to term infants indicate a maturing hypothalamic-pituitary-thyroid axis. Clinicians need thyroid hormone reference intervals that also vary by postmenstrual age to aid the evaluation of sick preterm infants who are at risk of a delayed hypothyroidism diagnosis that can be missed on the initial newborn screen. This study provides one of the largest samples of thyroid-stimulating hormone and free thyroxine data to establish reference intervals in preterm infants. Clinicians may utilize the identified postmenstrual age-based reference intervals to inform follow-up thyroid testing in preterm infants at several weeks postnatal age.

Objectives Thyroid dysfunction in pregnancy is associated with increased risk of adverse outcomes to mother and child. Trimester-specific reference intervals for thyroid function tests are not routinely provided by clinical laboratories. In this study, we present first- and second-trimester-specific reference intervals in a US population for thyroid-stimulating hormone (TSH), free thyroxine (FT4), total thyroxine (T4), and total triiodothyronine (T3) measured on Roche analyzers. Methods We used patient samples from first- and second-trimester prenatal screening. Samples were limited to singleton pregnancies and negative screening results for thyroid peroxidase and thyroglobulin antibodies. Analytes (TSH, FT4, T4, and T3) were measured on a Roche Modular e170 then verified on a Roche cobas e801. Results The reference intervals established on the e170 and verified on the e801 for the first trimester were 0.16 to 2.82 mIU/L for TSH, 12.0 to 18.5 pmol/L for FT4, 62.8 to 177.9 nmol/L for T4, and 1.5 to 3.4 nmol/L for T3. The reference intervals for the second trimester were 0.40 to 3.62 mIU/L for TSH, 10.2 to 16.6 pmol/L for FT4, 66.6 to 176.0 nmol/L for T4, and 1.56 to 3.6 nmol/L for T3. Conclusions This is the first report of trimester-specific reference intervals for thyroid function tests on Roche analyzers in the United States, and it is consistent with worldwide reports.

Background Dronabinol is used to treat a variety of conditions, including loss of appetite in people with AIDS and severe nausea and vomiting caused by cancer chemotherapy. Its therapeutic potential for pain management is now being explored in specific populations. Monitoring dronabinol compliance is challenging because its active ingredient, Δ-9-tetrahydrocannabinol (THC), is also present in cannabis. We developed a rapid LC-MS/MS assay with minimal specimen preparation to quantitate 11 cannabinoids in urine. Using this assay coupled with urine samples from normal controls, cannabis, and dronabinol users, we show the ability to differentiate cannabis from dronabinol use. Methods Residual clinical urine samples from 55 cannabinoid positive subjects and 31 negative controls, as well as prospective samples from 5 patients receiving dronabinol therapy were obtained for analysis. Results In the dronabinol group, only the THC metabolites 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-Δ-9-tetrahydrocannabinol (THC-OH) were detected. Minor cannabinoids were detected in 91% of cannabis group samples and their detection was more frequent in samples with increased THC metabolite concentrations. Of minor cannabinoids evaluated, cannabigerol (CBG) and cannabidiol (CBD) had the greatest sensitivity in detecting cannabis use. Conclusions This method has a high sensitivity for the detection of cannabis use with implications for evaluating dronabinol compliance.

Background Benzodiazepines (BZDs) are central nervous system depressants that are prescribed to prevent seizures, manage anxiety, or help sleep. When misused, BZDs can lead to addiction and sometimes cause death. Measurement of BZDs in urine is used to identify their use, especially in pain management settings. LC-MS/MS is preferred for these measurements because of its high sensitivity and specificity. Here, we report an LC-MS/MS assay for measuring 7 BZDs and metabolites in urine. Methods Urine sample was incubated at 60 °C for 30 min after addition of internal standards and a β-glucuronidase solution. After centrifugation, the supernatant was diluted with methanol and water before being injected onto a C18 analytical column in an LC-MS/MS system for quantification. The analytical time between injections was 4.35 min. The analytes included 7-aminoclonazepam, α-hydroxyalprazolam, α-hydroxytriazolam, oxazepam, lorazepam, nordiazepam, and temazepam. Results The lower limit of quantification ranged from 30 ng/mL to 50 ng/mL with an analytical recovery >80% for all 7 analytes. Total CV was 0.96) with the GC-MS method. Conclusions The LC-MS/MS assay allows accurate and precise measurement of 7 BZDs and metabolites in a single analytical run with a short analytical run time and broad measuring ranges.

Background A transition ion ratio (TIR) is the ratio of one fragment over another from the same precursor and is frequently monitored in liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for analyte identification. The Clinical and Laboratory Standards Institute (CLSI) C50-A guidelines give a static percent allowable TIR deviation based on the TIR level. Anecdotally, we observed failures of these rules for some of our LC-MS/MS assays. We determined what parameters may affect TIRs in a clinical setting and whether TIR variations may be analyte, matrix, instrument service, and/or concentration dependent. Methods Data was collected from the validation and selected periods after implementation for urine benzodiazepines (7 analytes) and plasma azole antifungals (6 analytes). TIRs for the calibrators and quality control materials on a Thermo TSQ™ Quantum Ultra from July 2016 to February 2017 for benzodiazepines in urine and Thermo TSQ™ Vantage from May 2016 to Oct 2016 for azoles in serum were monitored. Results The statistically significant day-to-day TIR shift ranged from 5.7 to 27.0% of the days studies for benzodiazepines and from 5.6 to 27.8% of the days studied for azoles excluding shifts caused by instrument services. Instrument service had significant impact on all benzodiazepines except oxazepam with p-values ranging from 1.79 × 10−6 to 1.53 × 10−39 and 4 of the 6 azoles (fluconazole, isavuconazole, voriconazole, and itraconazole) with (p from 7.89 × 10−3 to 1.98 × 10−12). Lorazepam, α-hydroxyalprazolam, and hydroxyitraconazole showed significant concentration dependent TIR variations. Conclusions TIR variations may be affected by instrument services, and can be concentration and analyte dependent. Instead of using a static percent deviation rule, establishment of TIR variation criteria for each analyte during test development and validation may provide a more useful tool for analyte identification.

The interference of bilirubin in both kinetic alkaline picrate creatinine assays (Jaffe) and enzymatic creatinine assays has been known and described in the literature (1, 2). Bilirubin negatively interferes with the Jaffe method, leading to underestimation of creatinine concentrations. Oxidation of bilirubin to biliverdin in alkaline solutions decreases the absorbance of both the creatinine picrate complex and bilirubin at the absorbance peak of 510 nm and increases the absorbance of biliverdin at the absorbance peak of 620 nm (3). However, bilirubin interference in the Jaffe method seems rather …

To develop a sensitive method for accurately measuring whole blood selenium and determining an appropriate reference interval for the local Cleveland population.The assay was developed and validated on an inductively coupled plasma mass spectrometry (ICP-MS) with a collision cell. Whole blood trace element free EDTA tubes were used to collect samples for the reference interval study (n=50). Samples were collected after at least 8h fast from healthy adults (76% females) with ages between 19 and 64yr. Whole blood aliquots (1mL) in acid washed cryogenic vials were stored at -70°C until analysis.The method passed the matrix effect, interference (except for Gd), and carryover tests. The method had a linear range of 0.2-7.1μmol/L with accuracies of 87.1-118.1%. The total assay imprecision (CV) was2.5% across the concentration levels tested. Comparison to another ICP-MS assay offered by an independent clinical lab yielded a Deming regression with a slope of 0.98, an intercept of 0.1μmol/L, a standard error of estimate of 0.1μmol/L, a correlation coefficient of 0.9846, and an average difference of 0.8%. The whole blood Se reference interval using a transformed parametric method was 2.2-3.5μmol/L.This whole blood Se ICP-MS methodology is sensitive and acceptable for patient testing.

Kidney disease is a major contributor to morbidity and mortality in the US, affecting 14% of the population. It is often referred to as a “silent epidemic” because the disease is often asymptomatic until the late stages and current clinical assays only detect it after a substantial portion of kidney capacity has been lost. As such, there is marked interest in kidney disease biomarkers that could identify the extent of kidney damage earlier in the pathological process. This may allow for interventions that could reduce or save the kidney from irreversible harm, thus reducing morbidity and mortality from this devastating disease. A clinically promising yet controversial biomarker for kidney disease, soluble urokinase-type plasminogen activator receptor (suPAR), was recently showcased in the journal Science (1). suPAR is the circulating form of a membrane protein …

BACKGROUND Health care data are increasing in volume and complexity. Storing and analyzing these data to implement precision medicine initiatives and data-driven research has exceeded the capabilities of traditional computer systems. Modern big data platforms must be adapted to the specific demands of health care and designed for scalability and growth. OBJECTIVE The objectives of our study were to (1) demonstrate the implementation of a data science platform built on open source technology within a large, academic health care system and (2) describe 2 computational health care applications built on such a platform. METHODS We deployed a data science platform based on several open source technologies to support real-time, big data workloads. We developed data-acquisition workflows for Apache Storm and NiFi in Java and Python to capture patient monitoring and laboratory data for downstream analytics. RESULTS Emerging data management approaches, along with open source technologies such as Hadoop, can be used to create integrated data lakes to store large, real-time datasets. This infrastructure also provides a robust analytics platform where health care and biomedical research data can be analyzed in near real time for precision medicine and computational health care use cases. CONCLUSIONS The implementation and use of integrated data science platforms offer organizations the opportunity to combine traditional datasets, including data from the electronic health record, with emerging big data sources, such as continuous patient monitoring and real-time laboratory results. These platforms can enable cost-effective and scalable analytics for the information that will be key to the delivery of precision medicine initiatives. Organizations that can take advantage of the technical advances found in data science platforms will have the opportunity to provide comprehensive access to health care data for computational health care and precision medicine research.

Background: Health care data are increasing in volume and complexity. Storing and analyzing these data to implement precision medicine initiatives and data-driven research has exceeded the capabilities of traditional computer systems. Modern big data platforms must be adapted to the specific demands of health care and designed for scalability and growth. Objective: The objectives of our study were to (1) demonstrate the implementation of a data science platform built on open source technology within a large, academic health care system and (2) describe 2 computational health care applications built on such a platform. Methods: We deployed a data science platform based on several open source technologies to support real-time, big data workloads. We developed data-acquisition workflows for Apache Storm and NiFi in Java and Python to capture patient monitoring and laboratory data for downstream analytics. Results: Emerging data management approaches, along with open source technologies such as Hadoop, can be used to create integrated data lakes to store large, real-time datasets. This infrastructure also provides a robust analytics platform where health care and biomedical research data can be analyzed in near real time for precision medicine and computational health care use cases. Conclusions: The implementation and use of integrated data science platforms offer organizations the opportunity to combine traditional datasets, including data from the electronic health record, with emerging big data sources, such as continuous patient monitoring and real-time laboratory results. These platforms can enable cost-effective and scalable analytics for the information that will be key to the delivery of precision medicine initiatives. Organizations that can take advantage of the technical advances found in data science platforms will have the opportunity to provide comprehensive access to health care data for computational health care and precision medicine research.

Immunosuppressant medications allow the transplantation of tens of thousands of allografts per year and consequently have great potential to decrease patient morbidity and mortality. However, some medications have great risk associated with over- and under-dosing leading to adverse effects or allograft rejection, respectively. This necessitates immunosuppressant therapeutic drug monitoring accomplished by immunoassay or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The former's accuracy can be hindered by metabolites of immunosuppressant medications, antibodies against these medications and heterophilic antibodies. Although LC-MS/MS has superior specificity which allows it to be less susceptible to interference, this methodology lacks standardization and the necessary throughput. Recent developments in LC-MS/MS quantitation, however, include patient-friendly sample submission as dried blood spots, higher sample throughput and commercialization. Here we critically review recent LC-MS/MS publications (January 2010 to July 2015) on the quantitation of cyclosporine A, tacrolimus, sirolimus and everolimus.

Summary Prostate cancer resistance to castration occurs because tumors acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signaling by the androgen receptor (AR) and the development of castration-resistant prostate cancer (CRPC)1–3. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly require enzymatic reactions by 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A) and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes4,5. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival6,7. We hypothesized that abiraterone is converted by an enzyme to the more active Δ4-abiraterone (D4A) that blocks multiple steroidogenic enzymes and antagonizes the androgen receptor (AR), providing an additional explanation for abiraterone’s clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3βHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive AR antagonism by D4A is comparable to the potent antagonist, enzalutamide. D4A also has more potent antitumor activity against xenograft tumors than abiraterone. Our findings suggest an additional explanation – conversion to a more active agent – for abiraterone’s survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment.

Background The frequency of variable hormonogenesis in patients with Cushing disease (CD) but without cyclical symptoms is unclear. Aim To assess the frequency of variable hormonogenesis in patients presenting with CD. Methods Over a 6-month period, patients with confirmed or suspected CD provided late-night salivary samples for up to 42 consecutive nights. Results Of 19 patients confirmed to have CD, 16 provided at least 7 consecutive salivary samples, and 13 provided at least 21; these 16 patients are the subjects of this report. Twelve patients had at least three peak and two trough levels of late-night salivary cortisol (LNSC) but in only two patients were strict criteria for cyclical hormonogenesis fulfilled; variation was assessed as random in the others. Eight patients had de novo CD, and eight had recurrent/persistent disease. All patients with recurrent/persistent CD had two or more normal results, and in four of these patients, >50% of LNSC were normal. In six patients with de novo disease with at least one normal LNSC level, the maximum levels ranged from 1.55 to 15.5 times the upper limit of normal. Conclusions Extreme fluctuations of cortisol production, measured by sequential LNSC, are common in CD. In newly diagnosed disease, this may only occasionally impair diagnostic ability, whereas in most patients with recurrent/persistent disease after pituitary surgery, LNSC is frequently within the reference range, with potential to cause diagnostic problems.

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

A 68-year-old man with a history of ischemic cardiomyopathy presented with decompensated heart failure and acute kidney injury. He was admitted to the coronary care unit for dopamine-assisted diuresis via a central line. Serum creatinine testing was performed using an enzymatic creatininase method (Roche cobas c701; Roche Diagnostics), revealing erratic creatinine concentrations …

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.

Objectives Chromium (Cr), a trace metal element, is implicated in diabetes and cardiovascular disease. A hypochromic state has been associated with poor blood glucose control and unfavorable lipid metabolism. Sensitive and accurate measurement of blood chromium is very important to assess the chromium nutritional status. However, interferents in biological matrices and contamination make the sensitive analysis challenging. The primary goal of this study was to develop a highly sensitive method for quantification of total Cr in whole blood by inductively coupled plasma mass spectrometry (ICP-MS) and to validate the reference interval in a local healthy population. Design and methods This method was developed on an ICP-MS with a collision/reaction cell. Interference was minimized using both kinetic energy discrimination between the quadrupole and hexapole and a selective collision gas (helium). Reference interval was validated in whole blood samples (n = 51) collected in trace element free EDTA tubes from healthy adults (12 males, 39 females), aged 19–64 years (38.8 ± 12.6), after a minimum of 8 h fasting. Blood samples were aliquoted into cryogenic vials and stored at − 70 °C until analysis. Results The assay linearity was 3.42 to 1446.59 nmol/L with an accuracy of 87.7 to 99.8%. The high sensitivity was achieved by minimization of interference through selective kinetic energy discrimination and selective collision using helium. The reference interval for total Cr using a non-parametric method was verified to be 3.92 to 7.48 nmol/L. Conclusion This validated ICP-MS methodology is highly sensitive and selective for measuring total Cr in whole blood.

Aim: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. Materials & methods: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. Results: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. Conclusion: These data support the strategy and the potential use of the biomarkers for PrCa., Lay abstract: The goal of this study was to use a novel strategy that combines biological outputs with Bayesian network learning to identify potential biomarkers for prostate cancer (PrCa). This methodology identified two proteins, filamin B and keratin-19, as potential biomarkers for PrCa. The network map also identified a direct linkage between filamin B and filamin A, which is a protein that has previously been identified as playing a role in PrCa etiology. The identified proteins were then validated by examining their levels in a panel of PrCa cell lines and in human plasma samples.

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.

Busulfan is used in myeloablative preparation regimens for hematopoietic bone marrow transplantation. Due to its narrow therapeutic range therapeutic drug monitoring of busulfan is recommended. In this study a fast and simple method for measuring busulfan in serum or plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed utilizing turbulent flow online extraction technology. Serum or plasma was mixed with acetonitrile containing d8-busulfan. After centrifugation the supernatant was injected onto a turbulent flow preparatory column then transferred to a C18 analytical column monitored by a tandem mass spectrometer set at positive electrospray ionization. The analytical cycle time was 4.0 min. The method was linear from 0.15 to 41.90 μmol/L with an accuracy of 87.9–103.0%. Inter- and intra-assay CVs across four concentration levels were 2.1–7.8%. No significant carryover or ion suppression was observed. No interference was observed from commercial control materials containing more than 100 compounds. Comparison with a well established LC–MS/MS method using patient specimens (n = 45) showed a mean bias 1.3% with Deming regression of slope 1.02, intercept −0.02 μmol/L, and a linear correlation coefficient 0.9883. The LC–MS/MS method coupled with turbulent flow online sample cleaning technology described here offers reliable busulfan quantitation in serum or plasma with minimum manual sample preparation and was fully validated for clinical use.

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important in maximizing the therapeutic response while minimizing the adverse effects. High-performance liquid chromatography (HPLC) is the most commonly used technique for this purpose. Recently, commercial monolithic columns were introduced, which consist of a single rod of fused silica or polymer. The objective of this work was to develop a simple and fast method to quantify 10 commonly measured AEDs or metabolites [carbamazepine, carbamazepine-10,11-epoxide, felbamate, lamotrigine, 10,11-dihydro-10-hydroxy-carbamazepine (active metabolite of oxcarbazepine), pentobarbital, phenobarbital, phenytoin, primidone, and zonisamide] in serum/plasma by HPLC using a reverse-phase monolithic column. Serum/heparin plasma (100 microL) was mixed with an internal standard solution (5-ethyl-5-p-tolylbarbituric acid in methanol, 250 microL). After centrifugation at 15,500 g for 10 minutes, 15 microL of supernatant was injected into a monolithic column. The analytes were eluted with an isocratic solution of 0.1 M, pH 6.5, phosphate buffer:methanol:acetonitrile (77:20:3), monitored at 210 nm. The chromatography time was 16 minutes. The method was linear from 0.4-4.9 to 21.2-190.9 microg/mL depending on the analytes with analytical recovery of 80%-114%. The inter- and intra-assay coefficients of variation were8% in 3 levels of serum-based controls for all the analytes. No significant carryover was observed. Commercial controls containing100 therapeutic drugs and common endogenous substances were tested and showed no interference. Comparison studies for 6 AEDs or metabolites were performed against commercial HPLC methods. Three AEDs were compared with Food and Drug Administration-approved immunoassays. All comparisons had R0.96 with slope ranging from 0.86 to 1.20. This is a simple and fast HPLC method suitable for measuring the 10 AEDs or metabolites. The use of the monolithic column resulted in increased sensitivity, better resolution, and a shorter analytical time compared with a regular C18 column.

Objectives Symmetric dimethylarginine (SDMA) is a catabolic product of arginine-methylated proteins and is an emerging biomarker for kidney function. A limited number of studies in selected populations have shown good correlation between SDMA and a few known markers of glomerular filtration rate (GFR). However, a comprehensive comparison of SDMA with all existing serum endogenous markers in a population with varied kidney function and against measured GFR is lacking. The objective of this study was to compare the correlations of SDMA, creatinine, cystatin C and their eGFR equations against GFR measured by iothalamate clearance in an adult population with varied kidney function. Design & methods Left-over serum and plasma specimens were collected from 40 adults with normal and reduced kidney function. GFR was measured using a radioactive iothalamate procedure. Creatinine and cystatin C were measured on Roche Cobas 8000. SDMA was measured by a published liquid chromatography-tandem mass spectrometry method. Results SDMA correlated highly with measured GFR (r = − 0.84), which was better than creatinine (r = − 0.70) but equivalent to cystatin C (r = − 0.86) and the eGFR equations [MDRD and CKD-EPI (separate and combined)]. Conclusions SDMA is a strong marker of kidney function and further studies are needed to establish an eGFR formula that includes it for widespread clinical use.

1,25-Dihydroxyvitamin D is the active form of vitamin D and plays a critical role in the maintenance of calcium and phosphorous metabolism of the human body. Measurement of 1,25-dihydroxyvitamin D in serum can aid in clinical diagnosis and/or management of renal disease, sarcoidosis, and rare inherited diseases. We present here an effective and accurate method for measuring 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) after immunoaffinity extraction. The MS/MS is operated in multiple reaction mode with positive electrospray. Quantification is based on peak area ratios of the analytes to respective deuterated internal standards. This method offered a linear range from 4.0 to 160.0 pg/mL with analytical recovery of 89.9-115.5 % for both 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2.

1,25-Dihydroxyvitamin D is the active form of vitamin D and plays a critical role in the maintenance of calcium and phosphorous metabolism of the human body. Measurement of 1,25-dihydroxyvitamin D in serum can aid in clinical diagnosis and/or management of renal disease, sarcoidosis, and rare inherited diseases. We present here an effective and accurate method for measuring 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) after immunoaffinity extraction. The MS/MS is operated in multiple reaction mode with positive electrospray. Quantification is based on peak area ratios of the analytes to respective deuterated internal standards. This method offered a linear range from 4.0 to 160.0 pg/mL with analytical recovery of 89.9-115.5 % for both 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2.

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 were6.0% 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.

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.

Background: Vitamin D is important to health and disease. Liquid chromatography-tandem mass spectrometry (LC-MSMS) is considered the most accurate technology for quantification of serum 25-hydroxyvitamin D (25OHD) which is the best biomarker for estimating vitamin D nutritional status. Methods: Serum was mixed with acetonitrile containing hexadeuterated 25-hydroxyvitamin D3 (d6-25OHD3) and centrifuged 10 min at 15,634×g. The supernatant was injected onto a turbulent flow preparatory column then transferred to a polar endcapped C18 analytical column. The mass spectrometer was set for positive atmospheric pressure chemical ionization. Results: The analytical cycle time was 5.5 min. Inter- and intra-assay CV for both analytes across three concentrations ranged from 3.8% to 14.2%. The method was linear from 3.0 to 283.6 nmol/L for 25-hydroxyvitamin D3 (25OHD3) and 4.6 to 277.9 nmol/L for 25-hydroxyvitamin D2 (25OHD2), with an accuracy of 88.7%–118.7% and 90.7%–100.3%, respectively. No carryover or ion suppression was observed. Comparison with a radioimmunoassay using patient specimens (n=527) showed a mean difference of 5.2%, and diagnostic agreement of 80.9% with Deming regression of slope 0.867, intercept 12.8, standard error of estimate (SEE) 17.4, and r=0.8425. Conclusions: The LC-MSMS method coupled with turbulent flow technology for serum 25OHD quantitation is rapid, efficient, and suitable for clinical testing. Clin Chem Lab Med 2009;47:1565–72.

Mycophenolic acid (MPA) is the primary active metabolite of the immunosuppressant mycophenolate mofetil. High performance liquid chromatography (HPLC) is commonly used for therapeutic drug monitoring (TDM) of MPA but requires batched runs. Recently, an enzyme inhibition assay (EIA) was approved for MPA TDM on random-access platforms using either serum or EDTA plasma. We evaluated the EIA on a Roche Integra 400 using serum and heparinized plasma in comparison with a validated HPLC method.: Heparinized plasma from leftover clinical samples on which MPA was ordered along with paired serum samples, drawn at the same time for other clinical tests, were used for the method comparison.: The EIA was linear from 3.1 to 44.0 μmol/L with an accuracy of 93.9%–107.1%. The intra- and inter-day variations were 0.5%–2.7% and 1.6%–2.1%, respectively. The limit of detection was 0.8 μmol/L and the limit of quantification was 3.1 μmol/L. The method showed a mean bias of 0.6 μmol/L (7.6%) in serum samples (3.1–34.1 μmol/L) vs. the HPLC method using paired plasma (n=229). Heparinized plasma (n=114) vs. serum showed a mean bias of –0.1 μmol/L (–1.6%) by the EIA.: The random-access EIA on Integra 400 is acceptable for clinical MPA TDM in either serum or heparinized plasma.Clin Chem Lab Med 2008;46:1281–4.