Evaluation of in vitro Skin Permeation of Clascoterone From Clascoterone Topical Cream, 1% (w/w).

Winlevi® (clascoterone) topical cream (1%, w/w) was approved by the U.S. FDA for the treatment of acne vulgaris in patients 12 years of age and older. The active ingredient, clascoterone, is not stable in physiological solutions and can hydrolyze to cortexolone at body temperature. Instability of clascoterone poses a significant challenge in accurately assessing the rate and extent of clascoterone permeation in vitro. Therefore, the purpose of this study was to develop an in vitro skin permeation test (IVPT) method, and a robust analytical method, that can minimize hydrolyzation of clascoterone during the study for quantification of clascoterone. Two IVPT methods, using either vertical diffusion cells or flow-through cells, were developed and compared to evaluate in vitro permeation of clascoterone from Winlevi. A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to monitor the level of clascoterone and cortexolone in the IVPT samples. The analytical method features a 2-min high-throughput analysis with good linearity, selectivity, and showed a lower limit of quantitation (LLOQ) of 0.5 ng/mL for both clascoterone and cortexolone. The in vitro skin permeation of clascoterone and cortexolone was observed as early as 2 h in both IVPT methods. A substantive amount of clascoterone was found to hydrolyze to cortexolone when using the vertical static diffusion cells with aliquot sampling. Conversely, degradation of clascoterone was significantly minimized when using the flow-through diffusion cells with fractional sampling. The data enhanced our understanding of in vitro permeation of clascoterone following topical application of the Winlevi topical cream, 1% and underscores the importance of IVPT method development and optimization during product development.

Salt-inducible kinase 1-CREB-regulated transcription coactivator 1 signalling in the paraventricular nucleus of the hypothalamus plays a role in depression by regulating the hypothalamic-pituitary-adrenal axis.

Elucidating the molecular mechanism underlying the hyperactivity of the hypothalamic-pituitary-adrenal axis during chronic stress is critical for understanding depression and treating depression. The secretion of corticotropin-releasing hormone (CRH) from neurons in the paraventricular nucleus (PVN) of the hypothalamus is controlled by salt-inducible kinases (SIKs) and CREB-regulated transcription co-activators (CRTCs). We hypothesised that the SIK-CRTC system in the PVN might contribute to the pathogenesis of depression. Thus, the present study employed chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression, various behavioural tests, virus-mediated gene transfer, enzyme-linked immunosorbent assay, western blotting, co-immunoprecipitation, quantitative real-time reverse transcription polymerase chain reaction, and immunofluorescence to investigate this connection. Our results revealed that both CSDS and CUMS induced significant changes in SIK1-CRTC1 signalling in PVN neurons. Both genetic knockdown of SIK1 and genetic overexpression of CRTC1 in the PVN simulated chronic stress, producing a depression-like phenotype in naive mice, and the CRTC1-CREB-CRH pathway mediates the pro-depressant actions induced by SIK1 knockdown in the PVN. In contrast, both genetic overexpression of SIK1 and genetic knockdown of CRTC1 in the PVN protected against CSDS and CUMS, leading to antidepressant-like effects in mice. Moreover, stereotactic infusion of TAT-SIK1 into the PVN also produced beneficial effects against chronic stress. Furthermore, the SIK1-CRTC1 system in the PVN played a role in the antidepressant actions of fluoxetine, paroxetine, venlafaxine, and duloxetine. Collectively, SIK1 and CRTC1 in PVN neurons are closely involved in depression neurobiology, and they could be viable targets for novel antidepressants.

Hippocampal miR-206-3p participates in the pathogenesis of depression via regulating the expression of BDNF.

As a widely-known neuropsychiatric disorder, the exact pathogenesis of depression remains elusive. MiRNA-206 (miR-206) is conventionally known as one of the myomiRs and has two forms: miR-206-3p and miR-206-5p. Recently, miR-206 has been demonstrated to regulate the biosynthesis of brain-derived neurotrophic factor (BDNF), a very popular target involved in depression and antidepressant responses. Here we assumed that miR-206 may play a role in depression, and various methods including the chronic social defeat stress (CSDS) model of depression, quantitative real-time reverse transcription PCR, western blotting, immuofluorescence and virus-mediated gene transfer were used together. It was found that CSDS robustly increased the level of miR-206-3p but not miR-206-5p in the hippocampus. Both genetic overexpression of hippocampal miR-206-3p and intranasal administration of AgomiR-206-3p induced not only notable depressive-like behaviors but also significantly decreased hippocampal BDNF signaling cascade and neurogenesis in naïve C57BL/6J mice. In contrast, both genetic knockdown of hippocampal miR-206-3p and intranasal administration of AntagomiR-206-3p produced significant antidepressant-like effects in the CSDS model of depression. Furthermore, it was found that the antidepressant-like effects induced by miR-206-3p inhibition require the hippocampal BDNF-TrkB system. Taken together, hippocampal miR-206-3p participates in the pathogenesis of depression by regulating BDNF biosynthesis and is a feasible antidepressant target.

Identification and profiling of targeted oxidized linoleic acid metabolites in rat plasma by quadrupole time-of-flight mass spectrometry.

Linoleic acid (LA) and LA-esters are the precursors of LA hydroperoxides, which are readily converted to 9- and 13-hydroxy-​octadecadienoic acid (HODE) and 9- and 13-oxo-​octadecadienoic acid (oxo ODE) metabolites in vivo. These four oxidized LA metabolites (OXLAMs) have been implicated in a variety of pathological conditions. Therefore, their accurate measurement may provide mechanistic insights into disease pathogenesis. Here we present a novel quadrupole time-of-flight mass spectrometry (Q-TOFMS) method for quantitation and identification of target OXLAMs in rat plasma. In this method, the esterified OXLAMs were base-hydrolyzed and followed by liquid-liquid extraction. Quantitative analyses were based on one-point standard addition with isotope dilution. The Q-TOFMS data of target metabolites were acquired and multiple reaction monitoring extracted-ion chromatograms were generated post-acquisition with a 10 ppm extraction window. The limit of quantitation was 9.7-35.9 nmol/L depending on the metabolite. The method was reproducible with a coefficient of variation of <18.5%. Mean concentrations of target metabolites in rat plasma were 57.8, 123.2, 218.1 and 57.8 nmol/L for 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE, respectively. Plasma levels of total OXLAMs were 456.9 nmol/L, which correlated well with published concentrations obtained by gas chromatography/mass spectrometry (GC/MS). The concentrations were also obtained utilizing a standard addition curve approach. The calibration curves were linear with correlation coefficients of >0.991. Concentrations of 9-HODE, 13-HODE, 9-oxoODE and 13-oxoODE were 84.0, 138.6, 263.0 and 69.5 nmol/L, respectively, which were consistent with the results obtained from one-point standard addition. Target metabolites were simultaneously characterized based on the accurate Q-TOFMS data. This is the first study of secondary LA metabolites using Q-TOFMS. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Virus-mediated decrease of LKB1 activity in the mPFC diminishes stress-induced depressive-like behaviors in mice.

As a highly prevalent neuropsychiatric disorder worldwide, the pathophysiology of depression is not yet fully understood and based on multiple factors among which chronic stress is critical. Numerous previous studies have shown the role of central mammalian target of rapamycin complex 1 (mTORC1) signaling in depression. However, so far it remains elusive by which way chronic stress down-regulates the activity of central mTORC1. Liver kinase b1 (LKB1) has been demonstrated to regulate the activity of the mTORC1 signaling cascade by phosphorylating AMP activated protein kinase (AMPK). Here, this study aimed to explore whether LKB1 participates in depression by regulating the downstream AMPK-mTORC1 signaling, and various methods including mouse models of depression, western blotting and immunofluorescence were used together. Our results showed that chronic stress significantly enhanced the expression of both phosphorylated LKB1 and total LKB1 in the medial prefrontal cortex (mPFC) but not the hippocampus. Furthermore, genetic knockdown of LKB1 in the mPFC fully reversed not only the depressive-like behaviors induced by chronic stress in mice but also the effects of chronic stress on the activity of AMPK and the mTORC1 system. Taken together, this study preliminarily suggests that LKB1 in the mPFC could be a feasible target for antidepressants. This study also provides support for the potential use of LKB1 inhibition strategies against the chronic stress-related neuropsychiatric disorders.

Hippocampal MSK1 regulates the behavioral and biological responses of mice to chronic social defeat stress: Involving of the BDNF-CREB signaling and neurogenesis.

Depression is one of the most common psychiatric diseases in the 21st century, while its pathogenesis is not yet fully understood. Currently, besides to the monoaminergic system, the brain-derived neurotrophic factor (BDNF)-cAMP response element-binding protein (CREB) signaling is one of the most attractive signaling pathways for treating depression. Mitogen and stress-activated kinase (MSK) 1 and 2 are nuclear proteins activated downstream of the ERK1/2 or p38 MAPK pathways, and it has been demonstrated that MSKs are involved in the BDNF-CREB signaling. Here we assumed that MSKs may play a role in depression, and various methods including the chronic social defeat stress (CSDS) model of depression, western blotting, immunofluorescence and virus-mediated gene transfer were used together. It was found that CSDS fully enhanced the expression of both phosphorylated MSK1 and total MSK1 in the hippocampus but not the medial prefrontal cortex (mPFC). CSDS did not influence the expression of phosphorylated MSK2 and total MSK2 in the two brain regions. Genetic over-expression of hippocampal MSK1 fully prevented not only the CSDS-induced depressive-like behaviors but also the CSDS-induced dysfunction in the hippocampal BDNF-CREB signaling and neurogenesis in mice, while genetic knockdown of hippocampal MSK1 aggravated the CSDS-induced depressive-like symptomatology in mice. Our results collectively suggest that although CSDS evidently enhances the activity of hippocampal MSK1, it is not a contributor to the CSDS-induced dysfunction in the brain but a defensive feedback regulator which protects against CSDS. Therefore, hippocampal MSK1 participates in the pathogenesis of depression and is a feasible and potential antidepressant target.

Ginsenoside Rh2 administration produces crucial antidepressant-like effects in a CUMS-induced mice model of depression.

INTRODUCTION: The most striking feature of depression is sadness and a loss of interest in activities, which represents a major cause of disability globally. Therefore, it is necessary to identify novel antidepressants for clinical practice. Ginsenoside Rh2 (Rh2) is one of the major bioactive ginsenosides that can be extracted from Panax ginseng and has been demonstrated to improve both memory and learning. The purpose of this study was to provide broad insight into the mechanisms underlying depression and gain greater insights into antidepressant therapy. METHODS: In this study, we first established an effective and feasible depression animal model of chronic unpredictable mild stress (CUMS) and behavioral testing was evaluated by the forced swim test (FST), the tail suspension test (TST) and the sucrose preference test. Following pretreatment with Rh2 (10 and 20 mg/kg), the immobility time of mice was reduced without affecting locomotor activity in both the FST and TST. Western blotting and immunofluorescence were used to investigate the activation of the hippocampal BDNF signaling pathway and hippocampal neurogenesis. RESULTS: Different concentrations of Rh2 significantly reduced depressive-like symptoms in CUMS-induced mice and downregulated the effects of the BDNF signaling cascade and neurogenesis in the hippocampus. Furthermore, the administration of K252a completely prevented the antidepressant-like activity of Rh2 in mice. CONCLUSION: The results indicated that Rh2 possesses the antidepression action via the positive regulation of the BDNF-TrkB pathway.

Simultaneous quantification of dexamethasone and 6ß-hydroxydexamethasone in rabbit plasma, aqueous and vitreous humor, and retina by UHPLC-MS/MS.

Aim: To develop and validate a fit for purpose method for the simultaneous determination of dexamethasone and its major metabolite, 6ß-hydroxydexamethasone, in rabbit plasma and ocular matrices to measure the in vivo release and distribution profile of dexamethasone from intravitreal implants. Materials & methods: An UHPLC-MS/MS system was employed to perform the bioanalysis. The method was validated according to the US FDA Bioanalytical Method Validation Guidance for Industry. Results & conclusion: The method was found to be fit-for-purpose for the described biological matrices and had a LLOQ of 0.1 ng/ml.

Xanthoceraside administration produces significant antidepressant effects in mice through activation of the hippocampal BDNF signaling pathway.

Current available antidepressants have various adverse reactions and slow pharmacodynamics, so it is necessary to find novel antidepressants for effective treatment. Xanthoceraside (XAN), a novel triterpenoid saponin extracted from the fruit husks of Xanthoceras sorbifolium Bunge, has anti-amnesic and neuroprotective properties. The purpose and significance of this study is to assess whether XAN has antidepressant effects in mice using the forced swim test (FST), tail suspension test (TST) and chronic unpredictable mild stress (CUMS) model of depression. The effects of XAN treatment on the hippocampal brain-derived neurotrophic factor (BDNF) signaling pathway and neurogenesis were examined. The antidepressant mechanism of XAN was explored using a BDNF inhibitor (K252a) and an anti-BDNF antibody. It was found that XAN administration significantly reversed the depressive-like behaviors of CUMS-treated mice. XAN treatment also significantly prevented the decreasing effects of CUMS on the hippocampal BDNF signaling and neurogenesis. The antidepressant effects of XAN in mice were blocked by both administration of K252a and anti-BDNF antibody. Collectively, these findings indicate that XAN possesses antidepressant effects in mice which are mediated by activation of hippocampal BDNF signaling pathway, thus providing the first evidence that XAN can be a potential antidepressant candidate.

Antidepressant-like activity of L-701324 in mice: A behavioral and neurobiological characterization.

Antidepressants currently used in clinical practice have limitations such as low efficacy, slow onset and various adverse reactions. It has become necessary to develop novel antidepressants beyond monoaminergic drugs. L-701,324 is a potent NMDA receptor antagonist, and the purpose of this study was to investigate the possible antidepressant effects of L-701,324 in mice. Here, various methods including the forced swim test (FST), tail suspension test (TST), chronic unpredictable mild stress (CUMS) model of depression, western blotting and immunofluorescence, were used together. A single injection of L-701,324 exhibited antidepressant-like potential in the FST and TST without affecting the locomotor activity of mice. Repeated injection of L-701,324 not only prevented CUMS-induced depressive-like behaviors in mice, but also ameliorated the downregulating effects of CUMS on the hippocampal BDNF signaling cascade and neurogenesis. Furthermore, K252a, a potent inhibitor of the BDNF system, fully blocked the antidepressant-like activity of L-701,324 in mice. K252a administration also abolished the activating actions of L-701,324 on the hippocampal BDNF signaling cascade and neurogenesis in CUMS-treated mice. Collectively, these data indicated that L-701,324 possesses antidepressant-like activity in mice, which was mediated, at least in part, by promoting the hippocampal BDNF system.

Antidepressant-like effects of 1-methylnicotinamide in a chronic unpredictable mild stress model of depression.

Depression is one of the most common psychiatric disorders, and there is strong demand for developing novel antidepressants with better efficacy and less adverse effects. 1-Methylnicotinamide (MNA) is a main metabolite of nicotinamide and has been demonstrated to possess biological effects in the brain. This study aimed to evaluate the antidepressant-like effects of MNA in mice, and the possible antidepressant mechanism was also determined. The forced swim test (FST), tail suspension test (TST), chronic unpredictable mild stress (CUMS) model of depression, western blotting method and K252a (a pharmacological inhibitor of the BDNF receptor) were used together in the present study. It was found that a single injection of MNA (100 and 200 mg/kg) displayed notable antidepressant-like potential in the FST and TST without affecting the locomotor activity of mice. Repeated administration of MNA (100 and 200 mg/kg) for 2 weeks fully reversed not only the CUMS-induced depressive-like symptoms in mice but also the CUMS-induced decrease in the hippocampal BDNF signaling pathway. Furthermore, the usage of K252a fully blocked the antidepressant-like effects of MNA in the FST, TST and CUMS model of depression. Collectively, MNA possess an antidepressant-like effect in mice which is mediated, at least in part, through promoting the hippocampal BDNF signaling pathway.

Hippocampal PPARα Plays a Role in the Pharmacological Mechanism of Vortioxetine, a Multimodal-Acting Antidepressant.

As a well-known multimodal-acting antidepressant, vortioxetine is thought to aim at several serotonin (5-HT) receptors and the 5-HT transporter. However, recently more and more proteins besides 5-HT are being reported to participate in the antidepressant mechanism of vortioxetine. As a widely known nuclear hormone receptor, peroxisome proliferator activated receptor α (PPARα) possesses transcriptional activity and is very important in the brain. Several reports have suggested that hippocampal PPARα is implicated in antidepressant responses. Here we speculate that hippocampal PPARα may participate in the antidepressant mechanism of vortioxetine. In this study, chronic unpredictable mild stress (CUMS), chronic social defeat stress (CSDS), behavioral tests, the western blotting and adenovirus associated virus (AAV)-mediated gene knockdown methods were used together. It was found that vortioxetine administration significantly reversed the inhibitory actions of both CUMS and CSDS on the hippocampal PPARα expression. Pharmacological blockade of PPARα notably prevented the antidepressant actions of vortioxetine in the CUMS and CSDS models. Moreover, genetic knockdown of PPARα in the hippocampus also significantly blocked the protecting effects of vortioxetine against both CUMS and CSDS. Therefore, the antidepressant effects of vortioxetine in mice require hippocampal PPARα.

Effect of herbal medicine Shenghua decoction on uterine bleeding after early medical abortion: A protocol for a systematic review and meta-analysis.

BACKGROUND: Excessive and prolonged uterine bleeding is an important obstacle for medical abortion to get popularized. Shenghua decoction (SHD) is widely used for treating uterine bleeding after early medical abortion. However, the clinical evidence is unclear. METHODS: Two researchers will dependently search literatures of SHD for the treatment of uterine bleeding after medical abortion from Web of Science, PubMed, Embase, and The Cochrane Library; traditional Chinese medicine databases; China National Knowledge Infrastructure (CNKI); Chinese Scientific Journal Database (VIP database); and Wan-Fang Database. These inclusive data of included studies will be conducted by RevMan V5.3 software. RESULTS: This systematic review and meta-analysis will provide a detailed summary of the current evidence related to the efficacy of SHD in treating uterine bleeding after early medical abortion, including the duration and volume of uterine bleeding, the medical abortion pain. CONCLUSION: This systematic review and meta-analysis will provide a detailed summary of the current evidence related to the efficacy of SHD in treating uterine bleeding after early medical abortion, REGISTRATION NUMBER:: PROSPERO CRD42020184465.

The Selective SIK2 Inhibitor ARN-3236 Produces Strong Antidepressant-Like Efficacy in Mice via the Hippocampal CRTC1-CREB-BDNF Pathway.

Depression is a widespread chronic medical illness affecting thoughts, mood, and physical health. However, the limited and delayed therapeutic efficacy of monoaminergic drugs has led to intensive research efforts to develop novel antidepressants. ARN-3236 is the first potent and selective inhibitor of salt-inducible kinase 2 (SIK2). In this study, a multidisciplinary approach was used to explore the antidepressant-like actions of ARN-3236 in mice. Chronic social defeat stress (CSDS) and chronic unpredictable mild stress (CUMS) models of depression, various behavioral tests, high performance liquid chromatography-tandem mass spectrometry, stereotactic infusion, viral-mediated gene transfer, western blotting, co-immunoprecipitation and immunofluorescence were used together. It was found that ARN-3236 could penetrate the blood-brain barrier. Repeated ARN-3236 administration induced significant antidepressant-like effects in both the CSDS and CUMS models of depression, accompanied with fully preventing the stress-enhanced SIK2 expression and cytoplasmic translocation of cyclic adenosine monophosphate response element binding protein (CREB)-regulated transcription coactivator 1 (CRTC1) in the hippocampus. ARN-3236 treatment also completely reversed the down-regulating effects of CSDS and CUMS on the hippocampal brain-derived neurotrophic factor (BDNF) system and neurogenesis. Moreover, we demonstrated that the hippocampal CRTC1-CREB-BDNF pathway mediated the antidepressant-like efficacy of ARN-3236. Collectively, ARN-3236 possesses strong protecting effects against chronic stress, and could be a novel antidepressant beyond monoaminergic drugs.

Simultaneous quantification of GM1 and GM2 gangliosides by isotope dilution tandem mass spectrometry.

OBJECTIVES: Gangliosides (GGs) are considered as diagnostic biomarkers and therapeutic targets and agents. The goal of this study was to develop a tandem mass spectrometry (MS/MS) method for the simultaneous measurement of both GM1 and GM2 gangliosides in human cerebrospinal fluid (CSF) samples in order to be able to determine their concentrations in patients with Tay-Sachs and Sandhoff disease and assess whether drugs or transplantation affect their concentrations. DESIGN AND METHODS: An API-4000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standards. To a 1.5 mL conical plastic Eppendorf centrifuge tube, 40 microL of human CSF sample was added and mixed with 400 microL of internal standard solution for deproteinization. After centrifugation, 100 microL of supernatant was injected onto a C-18 column. After a 2.5 min wash, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode. RESULTS: The within-day coefficients of variation were <3% for GM1 and <2% for GM2 and the between-day coefficients of variation were <5% for both GM1 and GM2 at all concentrations tested. Accuracy ranged between 98% and 102% for both analytes. Good linearity was also obtained within the concentration range of 10-200 ng/mL (6.5-129.3 nmol/L) for GM1 and 5-100 ng/mL (3.6-72.3 nmol/L) for GM2 (r> or =0.995). CONCLUSIONS: A new simple, accurate, and fast isotope dilution tandem mass spectrometry method was developed for the simultaneous quantification of GM1 and GM2 gangliosides in a small amount of human CSF. Concentrations were measured in "normal" CSF and in CSF from patients with Tay-Sachs disease.

Ex vivo expansion and transplantation of hematopoietic stem/progenitor cells supported by mesenchymal stem cells from human umbilical cord blood.

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45-55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

Simultaneous quantification of free triiodothyronine and free thyroxine by isotope dilution tandem mass spectrometry.

OBJECTIVES: This study was designed to improve our previously developed tandem mass spectrometry (MS/MS) method for free thyroxine (FT4) by enhancing sensitivity and permitting simultaneous measurements of both free triidothyronine (FT3) and FT4 using a smaller plasma/serum sample. DESIGN AND METHODS: An API-5,000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standard, T4-d(5). Four hundred microliters of human plasma/serum was filtered through a Centrifree YM-30 ultrafiltration device by centrifugation, and 450 microL of internal standard in methanol was then added to 150 microL of ultrafiltrate for deproteinization. After centrifugation, 500 microL of supernatant was diluted with 400 microL of distilled de-ionized water and a 650 microL aliquot was injected onto a C-18 column. After washing, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode. RESULTS: The within-day and between-day coefficients of variation (CVs) were

Tandem mass spectrometry improves the accuracy of free thyroxine measurements during pregnancy.

OBJECTIVE: Pregnancy is a time of rapidly changing demands on the thyroid axis, and knowledge of thyroid hormone levels, especially during the first trimester, is important for ensuring maternal and fetal health. The thyroid hormone assays currently in use become more inaccurate at extremes of binding protein concentrations and when heterophilic antibodies are present. Pregnancy is characterized by both these conditions, making accurate determination of free thyroid hormone levels by conventional direct analog immunoassay methods difficult. The objective of this study was to characterize the performance of a novel tandem mass spectrometric assay for free thyroxine during the physiologic conditions of pregnancy. DESIGN: Healthy women without a history of thyroid abnormalities were recruited from the obstetrics and gynecology and endocrinology clinics of a university medical center and their thyroid status was monitored. Free thyroxine levels were assessed by both immunoassay and tandem mass spectrometry during the course of their pregnancy. Serum thyrotropin levels were also measured. The distributions of free thyroid concentrations obtained by the two assays were compared. MAIN OUTCOME: The tandem mass spectrometry and immunoassay values did not correlate well with each other. However, tandem mass spectrometry values correlated well with the current gold standard equilibrium dialysis values. Moreover, the good agreement between equilibrium dialysis and tandem mass spectrometry was maintained across all weeks of gestation. CONCLUSIONS: We conclude that tandem mass spectrometry has a superior performance to immunoassay for the measurement of free thyroxine during pregnancy. Furthermore, it is ideally suited to generating trimester-specific reference intervals for free thyroxine levels. Future studies will determine if it is a better assay to use in most clinical circumstances.