Deuterium-labeled Internal Standards Research Articles

SLIM-Based High-Resolution Ion Mobility Reveals New Structural Insights into Isomeric Vitamin D Metabolites and their Isotopologues.

Testing for vitamin D deficiency remains a high-volume clinical assay, much of which is done using mass spectrometry-based methods to alleviate challenges in selectivity associated with immunoassays. Ion mobility-mass spectrometry (IM-MS) has been proposed as a rapid alternative to traditional LC-MS/MS methods, but understanding the structural ensemble that contributes to the ion mobility behavior of this molecular class is critical. Herein we demonstrate the first application of high-resolution Structures for Lossless Ion Manipulations (SLIM) IM separations of several groups of isomeric vitamin D metabolites. Despite previous IM studies of these molecules, the high resolving power of SLIM (Rp ∼ 200) has revealed additional conformations for several of the compounds. The highly similar collision cross sections (CCS), some differing by as little as 0.7%, precluded adequate characterization with low-resolution IM techniques where, in some cases, wider than expected peak widths and/or subtle shoulders may have hinted at their presence. Importantly, these newly resolved peaks often provided a unique mobility that could be used to separate isomers and provides potential for their use in quantification. Lastly, the contribution of isotopic labeling to arrival time distribution for commonly used 13C- and deuterium-labeled internal standards was explored. Minor shifts of ∼0.2-0.3% were observed, and in some instances these shifts were specific to the conformer being measured (i.e., "closed" vs "open"). Accounting for these shifts is important during raw data extraction to ensure reproducible peak area integration, which will be a critical consideration in future quantitative applications.

High-Throughput Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Method Validation for the Estimation of Atorvastatin and Active Metabolites in Human Plasma

A highly selective, sensitive, rugged, and rapid ultra high-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) is optimized and validated for reliable quantification of atorvastatin (ATR) and its active metabolites, 2-hydroxy atorvastatin (2-ATR) and 4-hydroxy atorvastatin (4-ATR) in human plasma using atorvastatin-D5 (ATR-D5), 2-hydroxy atorvastatin-D5 (2-ATR-D5), and 4-hydroxy atorvastatin-D5 (4-ATR-D5) as deuterium-labeled internal standards (ISTDs), respectively. Isocratic mode chromatographic separation was used with a reverse-phase C18 Symmetry Shield (150 × 4.6 mm, 5.0 μm) column and a mobile phase of acetonitrile:2 mM ammonium formate (pH-3.0) [65:35%v/v] at a flow rate of 0.7 mL/min. Electrospray ionization technique with positive ion mode polarity was applied to achieve the best signal intensity and stable response. Solid-phase extraction by direct elution method was applied to extract the drugs from the plasma sample. The calibration curve range was validated from a concentration range of 0.500-250 ng/mL for ATR and 2-ATR and 0.200-20 ng/mL for 4-ATR. The within-batch and between-batch precision and accuracy were found to be consistent and reproducible for all the analytes across the validation. Extraction recoveries were >80% for all analytes and ISTDs. All peaks of analytes and the respective ISTDs were eluted within 5.2 min. In this validated method, selective multivariate analytical approaches were utilized such as best fit linearity range for different strength formulations, preventive measures for in vivo and ex vivo autodegradation of metabolites, and shorter analysis time. This validated method can be useful for challenging quantification of ATR and its active metabolites for therapeutic drug monitoring and in high-throughput clinical study sample analysis.

Ultrafast liquid chromatography-tandem mass spectrometry determination of donepezil in human plasma: application to a bioequivalence study.

A liquid chromatography equipped with tandem mass spectrometric method using multi-stage flow rates was developed for the determination of donepezil in human plasma to support a randomized, crossover bioequivalence (BE) study in which healthy volunteers each received a single oral dose of the reference and test formulations of 10 mg donepezil hydrochloride. This integrated liquid chromatography with tandem mass spectrometry (LC-MS/MS) system with electrospray ionization and a deuterium-labeled internal standard (IS) were employed for the positive multiple-reaction-monitoring (MRM) analyses. The baseline separation using a high-resolution monolithic column under gradient and flexible flowrate conditions between donepezil and multiple interfering peaks from the extracted quality control, calibration standard and study plasma samples following simple protein precipitation extraction procedures was accomplished within 1.5 minutes. The ultrafast monolithic column performance in terms of chromatographic separation efficiency, peak asymmetry and resolution and retention time reproducibility was found to be sustainable. The linear dynamic range was detected over a concentration range of 0.2–50 ng/mL. The intra- and inter-day assay accuracy and precision were within 15% for the analyte in individual biological fluids. A positive correlation coefficient (r) greater than 0.995 for donepezil concentrations in study plasma samplers measured by the proposed and the other validated LC-MS/MS methods in support of a bioequivalence study was observed.

Determination of buprenorphine, norbuprenorphine, naloxone, and their glucuronides in urine by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), naloxone (NAL), and their glucuronide conjugates BUP-G, NBUP-G, and NAL-G in urine samples was developed. The method, omitting a hydrolysis step, involved non-polar solid-phase extraction, liquid chromatography on a C18 column, electrospray positive ionization, and mass analysis by multiple reaction monitoring. Quantification was based on the corresponding deuterium-labelled internal standards for each of the six analytes. The limit of quantification was 0.5μg/L for BUP and NAL, 1μg/L for NAL-G, and 3μg/L for NBUP, BUP-G, and NBUP-G. Using the developed method, 72 urine samples from buprenorphine-dependent patients were analysed to cover the concentration ranges encountered in a clinical setting. The median (maximum) concentration was 4.2μg/L (102 μg/L) for BUP, 74.7μg/L (580 μg/L) for NBUP, 0.9μg/L (85.5μg/L) for NAL, 159.5μg/L (1370 μg/L) for BUP-G, 307.5μg/L (1970 μg/L) for NBUP-G, and 79.6μg/L (2310 μg/L) for NAL-G.

An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

Effects of putrefaction on the quantitative analyses of diphenhydramine in blood and tissues: model experiments by the routine matrix-matched calibration method and standard addition method with different internal standards

The purpose of this study was to elucidate the effect of putrefaction on the quantitative analyses of diphenhydramine in blood and tissues by the routine matrix-matched calibration (MMC) method and the standard addition (SA) method for comparison. Putrefied samples were prepared by leaving pig blood, liver and muscle at room temperature. Toxic amounts of diphenhydramine and six internal standards (ISs) having different similarities were added to the prepared samples, and the drug was extracted with a liquid-liquid extraction method. The concentrations of diphenhydramine were determined by liquid chromatography–tandem mass spectrometry. The accuracies and precisions of the obtained quantitative values were compared in each quantification method. The results were also verified using human blood and tissues obtained at the time of autopsy. In the MMC method, the deuterium-labeled IS showed good accuracies and precisions for putrefied samples. On the other hand, the concentrations of diphenhydramine using some non-isotopic ISs were found to deviate significantly from the true value. The obtained results showed that the MMC method using fresh blood calibration curve with isotope-labeled IS could accurately measure drug concentrations in putrefied blood and tissues. If the isotope-labeled compound is not available, the quantification by the MMC method seems effective using several ISs with high similarity. This kind of study on the effects of putrefaction on forensic analyses has not been reported to our knowledge.

Fingerstick Plasma Drug Testing of Chronic Pain Patients: Comparison of Paired Fingerstick Plasma and Urine Specimens

Aim A clinical study was conducted to evaluate fingerstick blood as a viable biological matrix for monitoring prescription and illicit drugs in a clinical setting on patients undergoing pain and addiction treatment. The current standard for monitoring patients’ medication use, misuse, and diversion is urine drug testing (UDT). Materials and Methods This study compared 632 paired urine and fingerstick blood specimens collected at three pain management clinics and one suboxone clinic for 35 drugs and/or metabolites. Plasma from the fingerstick blood was used for the analysis. The urine and plasma specimens were analyzed by validated liquid chromatography–tandem mass spectrometry (LC-MS-MS) procedures. The urine cutoff used by most pain testing laboratories were used to identify positive and negative drugs in urine. Limit of quantitation was used to identify positive and negative drugs in plasma. Drugs and/or metabolites were quantified in both urine and plasma using deuterium-labeled internal standards. Results Results were tabulated for urine and plasma specimens for data analysis. The results showed that 8.7% of plasma specimens detected more drugs compared to the corresponding urine specimens, and 2.2% of the urine specimens detected a drug that was negative in the corresponding plasma specimen. Overall 89.1% of the specimens had complete agreement between urine and plasma specimens for detection. The observed Cohen’s Kappa value for overall drug detection was 0.96 an “almost perfect” agreement as characterized by Landis and Koch. Conclusion Based on the observed data, the authors conclude that plasma collected from fingerstick blood is a better matrix to monitor patients currently prescribed pain medications or patients currently undergoing medication-assisted opioid treatment compared to urine drug testing.

Limitations of deuterium-labeled internal standards for quantitative electrospray ionization mass spectrometry analysis of fatty acid metabolites.

The electrospray ionization mass spectrometry (ESI-MS) methodology often shows poor ionization reproducibility in the analysis of biological samples. Therefore, normalization of the measured peak intensities is essential. It is believed that quantitative data with high reproducibility can be obtained by adding a constant amount of an internal standard (IS) material labeled with stable isotopes to each sample, thus allowing the correction of the quantitative value of the target compound by that of the IS. We investigated whether the presence or absence of a labeled IS improves the accuracy of these quantitative values. Triple quadrupole MS coupled with liquid chromatography was used to analyze fatty acid metabolites in biological samples as target compounds. Two independent systems were used to provide a measure of reproducibility in two different laboratories. Data having poor reproducibility in the raw peak areas were efficiently normalized using the IS, but, crucially, the IS method using stable isotopes was not always necessary. In some cases, the reproducibility was relatively good even without using the IS. In a contaminant matrix, the MS response behavior of the target compound and its stable isotope-labeled material was complicated. Since ion suppression by matrix contaminants was dependent on the concentration of the target compound, the added amounts of the ISs were also important, Furthermore, an equivalent normalization effect was obtained by using a pooled quality control sample as an external standard, thus obviating the need for labeled IS samples, which are often expensive and sometimes not commercially available. Our results raise the question as to whether the quantitative method using stable-isotope-labeled ISs is always necessary and beneficial. However, the results obtained in this study cannot be generalized because only fatty acid metabolites were examined using ESI-MS and only a highly substituted deuterium-labeled IS was used.

MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (MP38)1 Apr 2020MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY Antonio Luigi Pastore*, Andrea Fuschi, Yazan Al Salhi, Gennaro Velotti, Lorenzo Capone, Alessia Martoccia, Pietro Paolo Suraci, Chiara Zerbinati, Luigi Iuliano, and Antonio Carbone Antonio Luigi Pastore*Antonio Luigi Pastore* More articles by this author , Andrea FuschiAndrea Fuschi More articles by this author , Yazan Al SalhiYazan Al Salhi More articles by this author , Gennaro VelottiGennaro Velotti More articles by this author , Lorenzo CaponeLorenzo Capone More articles by this author , Alessia MartocciaAlessia Martoccia More articles by this author , Pietro Paolo SuraciPietro Paolo Suraci More articles by this author , Chiara ZerbinatiChiara Zerbinati More articles by this author , Luigi IulianoLuigi Iuliano More articles by this author , and Antonio CarboneAntonio Carbone More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000887.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cholesterol is the main lipid component of sperm cell that is essential for sperm membrane fluidity, capacitation, and acrosomal reaction. Recent data obtained in bovine sperm showed that sperm capacitation is associated with the formation of oxysterols, oxidized products of cholesterol. The main objective of the present study was to identify and quantify, for the first time, the different species of oxysterols in human semen from normozoospermic, oligoasthenoteratozoospermic and asthenozoospermic patients. The secondary aim was to investigate the potential role of oxysterols in sperm pathophysiology. METHODS: To investigate the profile of human sperm oxysterols in subjects with normal and altered sperm characteristics, we recruited 150 consecutive subjects referring at the Centre of Andrology of Department of Urology for analysis of seminal fluid between March 2017 and June 2018. In order to investigate the possible correlation to oxidative stress and semen oxysterols profile, we included a group of patients with varicocele given the demonstrated relationship between varicocele and oxidative stress. Semen analysis was assessed by light microscope according to World Health Organization guidelines (WHO, 2010). Oxysterols were determined by GC–MS using deuterium-labelled internal standards as described by Dzeletovic et al. RESULTS: Complete oxysterol profile was obtained in 134 human semen of normozoospermic, oligoasthenoteratozoospermic, asthenozoospermic and varicocele patients. Oxysterols analyzed included seven autoxidation- and five enzymatically-generated oxysterols. Among the 12 oxysterols analyzed, 25-hydroxycholesterol (25-HC) resulted the most abundant oxysterol in normozoospermic subjects, and turned out to be the only one that differed significantly (p<0.0001) among the 4 groups. It was higher in the normozoospermic group (21.63±18.47 ng/mL, mean±SD) than oligoasthenoteratozoospermic (2.59±2.93, ng/mL, mean±SD), asthenozoospermic (5.59±3.17) and varicocele (13.48±11.81, ng/mL, mean±SD) groups. Furthermore, 25-HC positively correlated with the spermatozoa number (r=0.72, p<0.0001). CONCLUSIONS: In conclusion, to the best of our knowledge, this is the first study providing evidence for the feasibility of detection and quantification of oxysterols in human semen samples. We found in spermatozoa the presence of cholesterol 25-hydroxylase and its preferential accumulation in the neck and the post acrosomal area. 25-HC was able to induce calcium ions and cholesterol depletion in the acrosomal region, in an extent comparable to that of a capacitating medium. This finding offers the opportunity to identify a new molecular player involved in pre-fertilization processes. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e575-e576 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Antonio Luigi Pastore* More articles by this author Andrea Fuschi More articles by this author Yazan Al Salhi More articles by this author Gennaro Velotti More articles by this author Lorenzo Capone More articles by this author Alessia Martoccia More articles by this author Pietro Paolo Suraci More articles by this author Chiara Zerbinati More articles by this author Luigi Iuliano More articles by this author Antonio Carbone More articles by this author Expand All Advertisement PDF downloadLoading ...

A validated UHPLC-MS method for tryptophan metabolites: Application in the diagnosis of multiple sclerosis

The simultaneous quantitative estimation of tryptophan (TRP) and its metabolites represents a great challenge because of their diverse chemical properties, e.g., presence of acidic, basic, and nonpolar functional groups and their immensely different concentrations in biological matrices. A short ultra high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) method was validated for targeted analysis of TRP and its 11 most important metabolites derived via both kynurenine (KYN) and serotonin (SERO) pathways in human serum and cerebrospinal fluid (CSF): SERO, KYN, 3-hydroxyanthranilic acid, 5-hydroxyindoleacetic acid, anthranilic acid, kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), xanthurenic acid, melatonin, picolinic acid (PICA), and quinolinic acid (QUIN). After selecting the “best” reversed-phase column and organic modifier, DryLab®4 was used to optimize the gradient time and temperature in chromatographic separation. To achieve absolute quantification, deuterium-labeled internal standards were used. Among all compounds, 3 were analyzed in derivatized (butyl ester) forms (3-HK, PICA, and QUIN) and the remaining 9 in underivatized forms. Validation was performed in accordance with the ICH and FDA guidelines to determine the intraday and interday precision, accuracy, sensitivity, and recovery. To demonstrate the applicability of the developed UHPLC–MS/MS method, the aforementioned metabolites were analyzed in serum and CSF samples from patients with multiple sclerosis (multiple sclerosis group) and those with symptomatic or noninflammatory neurological diseases (control group). The concentration of QUIN dramatically increased, whereas that of KYNA slightly decreased in the multiple sclerosis group, resulting in a significantly increased QUIN/KYNA ratio and significantly decreased PICA/QUIN ratio.

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma.

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid-liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID-LC-MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. Graphical abstract Selected ion chromatograms obtained by liquid chromatography-tandem mass spectrometry with a C18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample.

An ultra-rapid drug screening method for acetaminophen in blood serum based on probe electrospray ionization-tandem mass spectrometry

Poisoning incidents caused by drugs, accidental ingestion of poisons, attempted suicide, homicide, and exposure to toxic compounds occur frequently every year across the globe. This raises the need to rapidly identify toxic agents in poisoned patients in a clinical emergency setting. In addition, determining drug/poison concentration is undoubtedly necessary to arrive at a toxicological treatment plan. The purpose of this study was to develop an ultra-rapid drug screening method for the clinical treatment of poisoning. Probe electrospray ionization (PESI), one of the ambient ionization techniques, is able to detect compounds from various biological materials almost directly. We applied the PESI technique to the rapid detection of acetaminophen (APAP). Blood serum samples were diluted 100-fold with 10 mM ammonium formate/ethanol (1:1 v/v) solution including deuterium-labeled internal standards (IS; APAP-d4). Only 10 μL of the diluted sample was used for measurement. The tandem mass spectrometer (MS/MS) equipped with a PESI was used in selected reaction monitoring mode for the quantitation of APAP; the measurement time was only 18 s. Transitions were set at 152 > 110 for quantitation, 152 > 65 for qualifier, and 156 > 114 for IS (APAP-d4). All measurements were conducted in positive mode. The calibration curve (1/x2) was linear over the range of 1.56–200 μg/mL (r2 = 0.998), and the limit of detection and quantitation were 0.37 μg/mL and 1.56 μg/mL, respectively. The accuracy (bias) and precision (RSD%) of the method were within an acceptable range (−0.15–2.8% and 2.3–6.1%, respectively) and matrix effect at 3 concentrations (95.1–104%) indicated that PESI-MS/MS is only slightly affected by matrices. In real forensic cases, quantitative values of APAP determined by the PESI-MS/MS were almost identical to those determined by the liquid chromatography-MS/MS method. Since PESI-MS/MS is a simple, reliable, and rapid determination method for toxic agents with virtually no need for blood serum pre-treatment, it would be highly suitable for poisoning cases in clinical emergency settings. In the future, a method for simultaneous rapid determination of multiple toxic agents will be developed.

Determination of Urinary Metabolites of the Emerging UV Filter Octocrylene by Online-SPE-LC-MS/MS.

Octocrylene (OC) is an emerging UV filter, which is used in the majority of sunscreens as well as other personal care products (PCP) and consumer products. Its presence in various environmental matrices has been reported. However, information on the internal OC exposure in humans is not available, due to the lack of appropriate biomarkers of exposure and analytical methods. Here, we describe a rugged, precise, and accurate analytical method for the determination of three OC metabolites (ester hydrolysis and alkyl chain oxidation products) in human urine by stable isotope dilution analysis. Urine samples are incubated with β-glucuronidase (E. coli K12) and then analyzed by liquid chromatography-electrospray ionization-triple quadrupole-tandem mass spectrometry with online turbulent flow chromatography for sample cleanup and analyte enrichment (online-SPE-LC-MS/MS). Syntheses of analytical standards, including deuterium-labeled internal standards, are also described. In a pilot study, we investigated the applicability of the metabolites as biomarkers of exposure in urine samples from the general population (n = 35). OC metabolites were detected in 91% of the samples, with the highest concentrations for three individuals having used sunscreen within 5 days prior to sample collection. We will apply the method in future human biomonitoring studies for OC exposure and risk assessment.

Description of analytical method and clinical utility of measuring serum 7-alpha-hydroxy-4-cholesten-3-one (7aC4) by mass spectrometry

BackgroundImbalance of bile acids (BA) homeostasis in the gastrointestinal tract can lead to chronic diarrhea or constipation when BA in the colon are in excess or low, respectively. Since both disturbances of bowel function can result from other etiologies, identifying BA imbalance is important to tailor treatment strategies. Serum concentrations of 7-alpha-hydroxy-4-cholesten-3-one (7aC4), a precursor in bile acid synthesis, reflect BA homeostasis. Here we describe a method to accurately measure serum 7aC4 and evaluate the clinical utility in patients with diarrhea or constipation phenotypes. MethodsSerum 7aC4 is measured after acetonitrile protein precipitation using C18 liquid chromatography, tandem mass spectrometry, and deuterium-labeled 7aC4 internal standard. Assay performance including linearity, precision, and accuracy was assessed using waste serum samples. The reference interval was established in healthy individuals without BA-altering conditions or medications. Clinical performance was assessed in patients with irritable bowel syndrome. ResultsThe method precisely and accurately measured 7aC4 in human serum from 1.4–338ng/mL with no ion suppression or interference from related 7-keto-cholesterol. Central 95th percentile reference interval was 2.5–63.2ng/mL. Lower serum 7aC4 was found in patients with constipation with sensitivity/specificity of 79%/55% compared to healthy controls. Higher 7aC4 was found in patients with bile acid diarrhea (BAD) compared to those without BAD with sensitivity/specificity of 82%/53%. ConclusionsWe have developed a sensitive and precise assay for measuring the concentration of 7aC4 in serum. The assay can be used to screen for diarrhea caused by bile acid malabsorption.

Quantitation of Rotundone in Grapefruit (Citrus paradisi) Peel and Juice by Stable Isotope Dilution Assay.

Aroma extract dilution analyses of the aromas of peels and juices of white and pink grapefruits revealed that rotundone, responsible for peppery, spicy, and woody odors, was detected for the first time at high flavor dilution factors of 256-1024. In both juices, rotundone was detected at the highest flavor dilution factor of 1024. Rotundone in grapefruits was quantitated by a stable isotope dilution assay with a newly synthesized deuterium-labeled internal standard, rotundone-d2,3: its levels were 2180 and 1920 ng/kg in white and pink grapefruit peels and 29.6 and 49.8 ng/kg in white and pink grapefruit juices, respectively. On the basis of these results, sensory analysis was performed to assess the effects of rotundone on a white grapefruit juice aroma reconstitute. This sensory analysis revealed that rotundone does not impart a woody odor or affect any of the existing attributes, but increases various attributes, thus confirming that rotundone is indispensable for the aroma of grapefruit juice.

Multiplex Tandem Mass Spectrometry Enzymatic Activity Assay for Newborn Screening of the Mucopolysaccharidoses and Type 2 Neuronal Ceroid Lipofuscinosis

We expanded the use of tandem mass spectrometry combined with liquid chromatography (LC-MS/MS) for multiplex newborn screening of seven lysosomal enzymes in dried blood spots (DBS). The new assays are for enzymes responsible for the mucopolysaccharidoses (MPS-I, -II, -IIIB, -IVA, -VI, and -VII) and type 2 neuronal ceroid lipofuscinosis (LINCL). New substrates were prepared and characterized for tripeptidyl peptidase 1 (TPP1), α-N-acetylglucosaminidase (NAGLU), and lysosomal β-glucuronidase (GUSB). These assays were combined with previously developed assays to provide a multiplex LC-MS/MS assay of 7 lysosomal storage diseases. Multiple reaction monitoring of ion dissociations for enzyme products and deuterium-labeled internal standards was used to quantify the enzyme activities. Deidentified DBS samples from 62 nonaffected newborns were analyzed to simultaneously determine (run time 2 min per DBS) the activities of TPP1, NAGLU, and GUSB, along with those for α-iduronidase (IDUA), iduronate-2-sulfatase (I2S), N-acetylgalactosamine-6-sulfatase (GALNS), and N-acetylgalactosamine-4-sulfatase (ARSB). The activities measured in the 7-plex format showed assay response-to-blank-activity ratios (analytical ranges) of 102-909 that clearly separated healthy infants from affected children. The new multiplex assay provides a robust comprehensive newborn screening assay for the mucopolysaccharidoses. The method has been expanded to include additional lysosomal storage diseases.

Matrix effect–corrected liquid chromatography/tandem mass-spectrometric method for determining acylcarnitines in human urine

Administration of pivalate-containing antibiotics decreases serum carnitine and increases urinary pivaloylcarnitine, resulting in hypocarnitinemia. Carnitine and acylcarnitines are important biomarkers in the diagnosis of carnitine deficiency, but the relationship between acylcarnitines and drug-induced hypocarnitinemia remains unclear. Quantification of acylcarnitines enables discovery of new biomarkers for prediction and diagnosis of drug-induced hypocarnitinemia. Here we describe a liquid chromatography/tandem mass-spectrometric method for simultaneously quantifying carnitine, 15 acylcarnitines, and cefditoren (the pivoxilated product of an antibiotic prodrug) in human urine. The matrix effect is corrected in 87.8–103% using deuterium-labeled internal standards (2H9-carnitine, 2H3-hexanoylcarnitine, and 2H3-stearoylcarnitine). The surrogate matrix method had an error of <13% in comparison with a standard addition method. Dynamic ranges were 0.1–100μmol/l for acylcarnitines and 0.3–300μg/ml for cefditoren. Both accuracy and precision were <19.7% at the lower limit of quantification and <14.8% for other quality controls. In an example application of this method, urine samples from eight healthy volunteers (five adults and three children) were analyzed, and individual differences were clearly observed.

Rapid analysis of bile acids in different biological matrices using LC-ESI-MS/MS for the investigation of bile acid transformation by mammalian gut bacteria.

Bile acids are important signaling molecules that regulate cholesterol, glucose, and energy homoeostasis and have thus been implicated in the development of metabolic disorders. Their bioavailability is strongly modulated by the gut microbiota, which contributes to generation of complex individual-specific bile acid profiles. Hence, it is important to have accurate methods at hand for precise measurement of these important metabolites. Here, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous identification and quantitation of primary and secondary bile acids as well as their taurine and glycine conjugates was developed and validated. Applicability of the method was demonstrated for mammalian tissues, biofluids, and cell culture media. The analytical approach mainly consists of a simple and rapid liquid-liquid extraction procedure in presence of deuterium-labeled internal standards. Baseline separation of all isobaric bile acid species was achieved and a linear correlation over a broad concentration range was observed. The method showed acceptable accuracy and precision on intra-day (1.42-11.07%) and inter-day (2.11-12.71%) analyses and achieved good recovery rates for representative analytes (83.7-107.1%). As a proof of concept, the analytical method was applied to mouse tissues and biofluids, but especially to samples from in vitro fermentations with gut bacteria of the family Coriobacteriaceae. The developed method revealed that the species Eggerthella lenta and Collinsella aerofaciens possess bile salt hydrolase activity, and for the first time that the species Enterorhabdus mucosicola is able to deconjugate and dehydrogenate primary bile acids in vitro.

Mass spectrometry-based ligand binding assays on adenosine A1 and A2A receptors.

Conventional methods to measure ligand-receptor binding parameters typically require radiolabeled ligands as probes. Despite the robustness of radioligand binding assays, they carry inherent disadvantages in terms of safety precautions, expensive synthesis, special lab requirements, and waste disposal. Mass spectrometry (MS) is a method that can selectively detect ligands without the need of a label. The sensitivity of MS equipment increases progressively, and currently, it is possible to detect low ligand quantities that are usually found in ligand binding assays. We developed a label-free MS ligand binding (MS binding) assay on the adenosine A1 and A2A receptors (A1AR and A2AAR), which are well-characterized members of the class A G protein-coupled receptor (GPCR) family. Radioligand binding assays for both receptors are well established, and ample data is available to compare and evaluate the performance of an MS binding assay. 1,3-Dipropyl-8-cyclopentyl-xanthine (DPCPX) and 4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a]-[1,3,5]triazin-5-yl)amino)ethyl)phenol (ZM-241,385) are high-affinity ligands selective for the A1AR and A2AAR, respectively. To proof the feasibility of MS binding on the A1AR and A2AAR, we first developed an MS detection method for unlabeled DPCPX and ZM-241,385. To serve as internal standards, both compounds were also deuterium-labeled. Subsequently, we investigated whether the two unlabeled compounds could substitute for their radiolabeled counterparts as marker ligands in binding experiments, including saturation, displacement, dissociation, and competition association assays. Furthermore, we investigated the accuracy of these assays if the use of internal standards was excluded. The results demonstrate the feasibility of the MS binding assay, even in the absence of a deuterium-labeled internal standard, and provide great promise for the further development of label-free assays based on MS for other GPCRs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-015-9477-0) contains supplementary material, which is available to authorized users.

Quantitative bile acid profiling by liquid chromatography quadrupole time-of-flight mass spectrometry: monitoring hepatitis B therapy by a novel Na(+)-taurocholate cotransporting polypeptide inhibitor.

A novel analytical approach for the targeted profiling of bile acids (BAs) in human serum/plasma based on liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) is presented. Reversed-phase chromatography enabled the baseline separation of 15 human BA species which could be readily detected by accurate mass analysis in negative ion mode. Blood proteins were removed by methanol precipitation in the presence of deuterium-labeled internal standards which allowed BA quantification in 50 μl plasma/serum. The assay was validated according to FDA guidance achieving quantification limits from 7.8 to 156 nM. Calibration curves prepared in charcoal-stripped serum/plasma showed excellent regression coefficients (R (2) > 0.997) and covered quantities from 7.8 to 10,000 nM depending on the analyzed species. Intra- and inter-day accuracy and precision were below 15 % for all analytes. Apparent extraction recoveries were above 97 %, and ion suppression rates were between 4 and 53 %. Mean BA level in serum/plasma from healthy volunteers ranged from 11 ± 4 nM (tauroursodeoxycholic acid) to 1321 ± 1442 nM (glycochenodeoxycholic acid). As a proof of concept, the assay was applied to plasma samples derived from a clinical phase I study of myrcludex B, a novel first-in-class virus entry inhibitor for the treatment of chronic hepatitis B and D. The results demonstrate that myrcludex-induced inhibition of the hepatic BA transporter Na(+)-taurocholate cotransporting polypeptide (NTCP) significantly affects plasma BA level. These observations provide novel insights into drug-induced metabolic responses and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials.