Cold-induced phase separation for the simple and reliable extraction of sex hormones for subsequent LC-MS/MS analysis.

Sex hormones, including androgens, estrogens, and progestogens, are important biomarkers for various diseases. Quantification of sex hormones is typically conducted by LC-MS/MS. At present, most methods require liquid-liquid extraction or solid phase extraction for sample preparation. However, these pretreatments are prone to compromise LC-MS/MS throughput. To improve on the current standard practices, we investigated cold-induced phase separation for sex hormone extraction. After protein precipitation with acetonitrile and adjusting the solution constitution with water, samples were stored at -30°C for 10 min to generate two distinct phases: an acetonitrile-rich layer on top of a water-rich layer. During this process, the hydrophobic sex hormones spontaneously separate into the upper layer. This simple and reliable cold-induced phase separation-based LC-MS/MS methodology was used here to simultaneously detect estrone, estradiol, estriol, testosterone, androstenedione, dehydroepiandrosterone, progesterone, and 17-hydroxyprogesterone in serum. Validation of this method indicated satisfactory performance, including acceptable linearity, accuracy, precision, and tractability. Compared with the mainstream liquid-liquid extraction-based method, this new method exhibits significant progress in throughput, which shortens the time cost of sample preparation from 90 to 40 min. We propose that this method can be an excellent alternative for sex hormone analysis in routine clinical laboratories.

Rapid Derivatization of Phenolic and Oxime Hydroxyl with Isonicotinoyl Chloride under Aqueous Conditions and Its Application in LC-MS/MS Profiling Multiclass Steroids.

Quantification of steroids possesses a crucial clinical value in early diagnosis and prognosis evaluation of various endocrine diseases. However, it is still challenging to realize feasible analysis of estrogens, androgens, progestogens, and corticoids within one single workflow. In this study, two derivatization reactions were newly designed for improvement: (1) acylation of phenolic hydroxyl on estrogens with isonicotinoyl chloride (INC) under the catalysis of 4-dimethylaminopyridine and (2) post-modification of oxime hydroxyl on hydroxylamine-pretreated ketosteroids with INC. Both reactions could conduct instantaneously at room temperature under aqueous conditions. Moreover, the resulting phenolic-INC and oxime-INC esters exhibited favorable MS responses. Through integrating these derivatization strategies with cold-induced phase separation technology, a feasible LC-MS/MS method was developed for simultaneous quantification of 15 multiclass steroids with proper sample consumption (50 µL serum), satisfying sensitivity (lower limit of quantitation at 0.01-5.00 ng/mL) and high throughput (40 min for sample-preparation). The practical applicability was tested by detecting 30 real samples from pregnant and non-pregnant women. The obtained results showed a good agreement with a previous validated methodology.

Ga-68 EDTA aerosols in evaluation of inhaled-particle deposition and clearance of obstructive pulmonary diseases: A pilot prospective study compared with Galligas.

PURPOSE: 68-gallium (Ga-68) ethylenediaminetetraacetic acid (EDTA) aerosols and Galligas were compared in evaluation of inhaled-particle deposition and clearance in volunteers with or without obstructive pulmonary diseases. METHODS: Nonsmoking healthy volunteers, healthy smokers, asthma patients and patients with chronic obstructive pulmonary disease (COPD) were recruited to undergo the dynamic lung ventilation positron emission tomography/computerized tomography (PET/CT) scans within two consecutive days. The inhaled particles were Ga-68-labelled carbon nanoparticles (Galligas, 30-60 nm in size) and Ga-68-labelled EDTA aerosols (1-2 µm in size), respectively. The volunteers' lung function parameters were measured for comparison. RESULTS: Central deposition and inhomogeneity of both tracers were negatively correlated with lung function parameters, including the ratio of forced expiratory volume at 1 second to forced vital capacity (FEV1 /FVC). The central or hilum deposition of Galligas, but not 68-gallium (Ga-68) EDTA, was negatively correlated with the maximal expiratory flow at 25%, 50% and 75% of the forced vital capacity. Compared with Galligas, Ga-68 EDTA aerosols were more concentrated in the central region in all groups except for the healthy nonsmokers. Ventilation inhomogeneity was more evident when using Ga-68 EDTA aerosols, especially in patients with COPD and asthma patients. In the healthy smokers, the central region accumulated more Ga-68 EDTA at 30 minutes after inhalation than immediately after inhalation. Ga-68 EDTA cleared faster in lungs than Galligas. CONCLUSIONS: Both Galligas and Ga-68 EDTA aerosols can be used for PET/CT lung ventilation scan. However, Ga-68 EDTA aerosols showed more advantages in diagnosis and evaluation of obstructive airway diseases by revealing the inhaled-particle deposition and clearance.

An LC/MS/MS method for analyzing the steroid metabolome with high accuracy and from small serum samples.

Analyzing global steroid metabolism in humans can shed light on the etiologies of steroid-related diseases. However, existing methods require large amounts of serum and lack the evaluation of accuracy. Here, we developed an LC/MS/MS method for the simultaneous quantification of 12 steroid hormones: testosterone, pregnenolone, progesterone, androstenedione, corticosterone, 11-deoxycortisol, cortisol, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, estriol, and estradiol. Steroids and spiked internal standards in 100 µl serum were extracted by protein precipitation and liquid-liquid extraction. The organic phase was dried by evaporation, and isonicotinoyl chloride was added for steroid derivatization, followed by evaporation under nitrogen and redissolution in 50% methanol. Chromatographic separation was performed on a reverse-phase PFP column, and analytes were detected on a triple quadrupole mass spectrometer with ESI. The lower limits of quantification ranged from 0.005 ng/ml for estradiol to 1 ng/ml for cortisol. Apparent recoveries of steroids at high, medium, and low concentrations in quality control samples were between 86.4% and 115.0%. There were limited biases (-10.7% to 10.5%) between the measured values and the authentic values, indicating that the method has excellent reliability. An analysis of the steroid metabolome in pregnant women highlighted the applicability of the method in clinical serum samples. We conclude that the LC/MS/MS method reported here enables steroid metabolome analysis with high accuracy and reduced serum consumption, indicating that it may be a useful tool in both clinical and scientific laboratory research.

Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a slow albeit progressive rare neoplastic disease featured with diffuse thin-walled cysts in lungs and angiomyolipomas in kidneys. LAM affects almost exclusively women and has one of the strongest gender predispositions of any extragenital human disease. Two forms of LAM present clinically, sporadic (S-LAM) and tuberous sclerosis complex-associated (TSC-LAM). TSC is an autosomal dominant genetic multisystems neoplastic disease. A high prevalence of LAM can be detected in adult female TSC patients. Tremendous progress has been made in our understanding and management of this rare disease. Both LAM and TSC are TSC2 or TSC1 mutated diseases that result in overactivation of the mechanistic target of rapamycin (mTOR) pathway. Sirolimus, an mTOR inhibitor, has been approved for LAM treatment in the United States and many other countries. Therapies targeting female sex hormones have shown preclinical efficacy in animal and cell culture-based experiments, but have not been properly investigated clinically. In this review, we summarize current recommendations in the diagnosis and treatment of LAM.

Lung function and systemic inflammation associated with short-term air pollution exposure in chronic obstructive pulmonary disease patients in Beijing, China.

BACKGROUND: Exposure to air pollution is associated with chronic obstructive pulmonary disease (COPD). However, findings on the effects of air pollution on lung function and systemic inflammation in Chinese COPD patients are inconsistent and scarce. This study aims to evaluate the effects of ambient air pollution on lung function parameters and serum cytokine levels in a COPD cohort in Beijing, China. METHODS: We enrolled COPD participants on a rolling basis from December 2015 to September 2017 in Beijing, China. Follow-ups were performed every 3 months for each participant. Serum levels of 20 cytokines were detected every 6 months. Hourly ambient pollutant levels over the same periods were obtained from 35 monitoring stations across Beijing. Geocoded residential addresses of the participants were used to estimate daily mean pollution exposures. A linear mixed-effect model was applied to explore the effects of air pollutants on health in the first-year of follow-up. RESULTS: A total of 84 COPD patients were enrolled at baseline. Of those, 75 COPD patients completed the first-year of follow-up. We found adverse cumulative effects of particulate matter less than 2.5 µm in aerodynamic diameter (PM2.5), nitrogen dioxide (NO2), sulfur dioxide (SO2) and carbon monoxide (CO) on the forced vital capacity % predicted (FVC % pred) in patients with COPD. Further analyses illustrated that among COPD patients, air pollution exposure was associated with reduced levels of serum eotaxin, interleukin 4 (IL-4) and IL-13 and was correlated with increased serum IL-2, IL-12, IL-17A, interferon γ (IFNγ), monocyte displacing protein 1 (MCP-1) and soluble CD40 ligand (sCD40L). CONCLUSION: Acute exposures to PM2.5, NO2, SO2 and CO were associated with a reduction in FVC % pred in COPD patients. Furthermore, short-term exposure to air pollutants increased systemic inflammation in COPD patients; this may be attributed to increased Th1 and Th17 cytokines and decreased Th2 cytokines.

LC-MS/MS determination of plasma catecholamines after selective extraction by borated zirconia.

For the first time, boronate affinity chromatography and metal oxide affinity chromatography mechanisms for cis-diol-compounds extraction are simultaneously realized on a single material, termed borated zirconia. This material was prepared by hydrolyzing zirconium butoxide with boric acid under non-aqueous environment. The diameter of formed particles is around 200 nm. By extracting catechol under different pH conditions or with phosphate ion competition, the dual affinity mechanisms of borated zirconia are confirmed. Benefiting from such unique feature, borated zirconia can function well under neutral condition. By using borated zirconia for dispersive solid phase extraction, specific capture of cis-diol-containing catecholamines, including epinephrine (E), norepinephrine (NE) and dopamine (DA), was achieved. A reliable LC-MS/MS method was established and validated for quantification of these target analytes in plasma samples after derivatisation with benzoyl chloride. The linear ranges are 0.010-0.200 ng·mL-1 for E and DA, and 0.050-1.000 ng·mL-1 for NE. The limits of quantification are 0.008, 0.020 and 0.004 ng·mL-1 for E, NE and DA respectively. By analyzing samples from healthy volunteers and schizophrenia patients, the plasma concentrations of E, NE and DA were found to be higher for the latter. Graphical AbstractSchematic representation of boronate combined metal oxide affinity chromatography (BMOAC) extraction of cis-diol catecholamines from human plasma by borated zirconia following with benzoyl chloride derivatization and LC-MS determination.

Role of Statins in Treatment and Prevention of Community-acquired Pneumonia:A Meta-analysis.

Objective To systemically evaluate the the role of statins in prevention and treatment of community-acquired pneumonia (CAP). Methods A computer-based searching was conducted in PubMed,EMbase,Cochrane Library,Wanfang,and CNKI database up to October 2016. Totally 21 eligible articles were retrieved. According to the Cochrane Handbook 5.0 or Newcastle-Ottawa Scale (NOS) quality evaluation criteria,two independent reviewers carried out literature screening,data retraction,and quality evaluation. Meta-analysis was conducted with RevMan 5.3 software. Results A total 1 007 765 CAP patients from 12 studies were divided into two groups:statin group (n=118 096) and non-statin group (n=889 669). Meta-analysis suggested that statin use was associated with decreased mortality of CAP (OR=0.67,95%CI:0.57-0.79). We further divided the studies into North America group and Europe group and found the heterogenicity of North America group was lower than that of Europe group,and the Meta-analysis of both group supported the association of statin use with decreased CAP mortality (OR=0.66,95%CI:0.62-0.67;OR=0.71,95%CI:0.55-0.92). To identify the effect of statin use on mechanical ventilation,we included three articles (n=123 645) for further analysis (statin group,n=23 796;non-statin group,n=99 849),and Meta-analysis suggested that statin use was associated with decreased requirement for mechanical ventilation (OR=0.74,95%CI:0.70-0.78). Four articles (n=127 060) were enrolled (statin group,n=24 121 and non-statin group,n=102 939) to analyze the effect of statin use on ICU admission,and Meta-analysis suggested that statin use was associated with decreased requirement for ICU admission (OR=0.85,95%CI:0.82-0.88). Eleven articles (n=2 124 849) (statin group,n=306 108;non-statin group,n=1 818 741) to evaluate the effect of statin use on risk of CAP,and Meta-analysis suggested that long-term use of statins decreased the risk of CAP,although there was no statistical difference (OR=0.85,95%CI:0.85-1.07); the above studies were divided into case-control studies and cohort studies,and the case-control studies revealed statins increased the risk of CAP (OR=1.12,95%CI:1.03-1.21),while the cohort studies supported the association of statin use with decreased CAP risk (OR=0.46,95%CI:0.44-0.49). Conclusions Statin use may decrease the CAP mortality and the requirement for mechanical ventilation or ICU admission. However,whether statin use can reduce the risk of pneumonia remains unclear.

Development of a UPLC-MS/MS method for routine therapeutic drug monitoring of aripiprazole, amisulpride, olanzapine, paliperidone and ziprasidone with a discussion of their therapeutic reference ranges for Chinese patients.

A highly feasible and reliable ultra-high performance liquid chromatography tandem mass spectrometry method was presented for therapeutic drug monitoring of five anti-schizophrenic drugs (amisulpride, olanzapine, aripiprazole, paliperidone and ziprasidone) simultaneously. To meet the requirements of practical clinical usage (easy handling, high throughput and cost effectiveness), the pretreatment process was simplified (only including protein precipitation and mobile phase dilution steps) and the UPLC separation cycle was set within 6 min. The whole methodology was carefully validated according to the latest international guidelines showing the excellent selectivity, accuracy, precision, applicability and stability. After a 10 month clinical application, a retrospective analysis of 253 positive samples was carried out to investigate conformance with the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie therapeutic reference range for Chinese patients. The results suggested good consistency for olanzapine, aripiprazole, paliperidone and ziprasidone, while for amisulpride, the plasma concentration level (445.2 ± 231.5 ng/mL) was relatively higher than the recommended range (100-320 ng/mL). We supposed that such phenomenon indicated the necessity of reconstructing a Chinese-specific therapeutic reference range for amisulpride treatment, which would be helpful to improve medication efficiency and safety for Chinese patients.

Advancing towards practice: A novel LC-MS/MS method for detecting retinol in dried blood spots.

BACKGROUND: To date, clinical laboratories face challenges in quantifying retinol from DBS samples. Disputes arise throughout the whole detection process, encompassing the storage condition, the release strategy as well as the selection of internal standards. METHODS: We incubated DBS with ascorbic acid solution. Then, retinol-d4 in acetonitrile was introduced to incorporate isotopic internal standard and promote protein precipitation. Afterward, sodium carbonate solution was added to ionize cytochromes (such as bilirubin), which amplified the difference of their hydrophobicity to retinol. Subsequently, cold-induced phase separation could be facilitated to separate retinol from the impurities. In the end, the upper layer was injected for LC-MS/MS analysis. RESULTS: By comparing the detected retinol content in whole blood and DBS samples prepared from the same volume, we confirmed the established pretreatment was capable to extract most of retinol from DBS (recovery >90 %). Thereafter, we verified that within DBS, retinol possessed satisfying stability without antioxidation. Indoor-light exposure and storage duration would not cause obvious degradation (<10 %). Following systematic validation, the established method well met the criteria outlined in the relevant guidelines. After comparing with detected DBS results to the paired plasma samples, 54 out of 60 met the acceptance limit for cross-validation of ±20 %. CONCLUSIONS: We realized precise quantification of retinol from one 3.2 mm DBS disc. By circumventing conventional antioxidation, liquid-liquid/solid-phase extraction and organic solvent evaporation, the pretreatment could be completed within 15 min consuming only minimal amounts of low-toxicity chemicals (ascorbic acid, acetonitrile, and sodium carbonate). We expect this contribution holds the potential to significantly facilitate the evaluation of patients' vitamin A status by using DBS samples in the future.

Associations Between Vitamin K and Suicide Attempts in Patients with Depression: A Case-Control Study.

Background: Hypovitaminosis K has been linked to depression and suicide, but epidemiological research is scarce. This study aimed to explore the association among vitamin K with depression and suicidal attempts. Methods: This was a retrospective cross-sectional study involving 146 cases with a history of suicidal attempts and 149 subjects without a lifetime history of suicidal attempts. The levels of thyroid hormones, lipid profile, inflammatory cytokines, and vitamins were measured. Results: Subjects who had suicidal attempts presented with a significant decrease in FT4, TC, vitamin D, and vitamin K but increased CRP levels. In these variables, vitamin K has a better diagnostic value for suicidal attempts in depressed patients, with a sensitivity of 0.842 and a specificity of 0.715. Correlation analysis suggested that vitamin K was significantly and positively related to FT4, TC, LDL, and sdLDL. Multivariate analysis showed that serum vitamin K level predicts suicidal attempts in depressive patients (OR = 0.614, P = 0.004, 95% CI 0.153-0.904). Moreover, a negative correlation between vitamin K and suicidal attempts was also noted for partial FT4, CRP, and vitamin D strata analysis. Conclusion: Our study suggests that low vitamin K levels were correlated with suicidal attempts in patients with depression, indicating that vitamin K deficiency might be a biological risk factor for depression.

Risk factors associated with severe adverse events in patients with relapsing polychondritis undergoing flexible bronchoscopy.

BACKGROUND: Patients with relapsing polychondritis (RP) sometimes experience upper airway collapse or lower airway stenosis, and bronchoscopy may provide a valuable typical image to confirm the diagnosis. This study aimed to identify potential risk factors associated with severe adverse effects during bronchoscopy. METHODS: We performed a retrospective cohort study of 82 consecutive patients with RP hospitalized at Peking Union Medical College Hospital between January 1, 2012 and December 31, 2022. Clinical features and disease patterns were compared among patients with RP undergoing bronchoscopy with or without severe adverse effects. Binary logistic regression analysis was performed to identify the associated risk factors. RESULTS: For patients with RP undergoing bronchoscopy with severe adverse effects, the forced vital capacity (FVC), forced vital capacity percent predicted values (FVC%), and peak expiratory flow were significantly lower (P = 0.001, P = 0.001, and P = 0.021, respectively) than those in the non-severe adverse effect subgroup. Binary logistic regression analysis revealed that low FVC% (odds ratio, 0.930; 95% confidence interval, 0.880-0.982; P = 0.009) was an independent risk factor for severe adverse events in patients undergoing bronchoscopy. CONCLUSIONS: Low FVC or FVC% suggests a high risk of severe adverse effects in patients with RP undergoing bronchoscopy. Patients with such risk factors should be carefully evaluated before bronchoscopy and adequately prepared for emergency tracheal intubation or tracheostomy.

TiO2-based solid phase extraction strategy for highly effective elimination of normal ribonucleosides before detection of 2'-deoxynucleosides/low-abundance 2'-O-modified ribonucleosides.

A novel TiO2-based SPE strategy was developed for eliminating normal ribonucleosides before mass spectrometry (MS) analysis of 2'-deoxynucleosides and 2'-O-modified ribonucleosides. The chromatographic research for the retention behavior of ribonucleosides and 2'-deoxynucleosides on TiO2 materials was investigated using TiO2 separation column. The results indicated a specific affinity interaction mechanism between TiO2 and cis-diol-containing ribonucleosides, and the interaction was proved effective even under a wide range of pH conditions and salt concentrations. Benefiting from these features, a TiO2-based solid phase extraction (SPE) method was developed for highly efficient elimination of RNA contamination from genomic DNA. Compared with the widely used enzymatic digestion method, the proposed TiO2-based SPE method showed much more efficiency for the removal of RNA as well as provided high recoveries for the 2'-deoxynucleosides. In addition, the sample processing time is dramatically shortened using the TiO2-based SPE method (~5 min) compared to the traditional enzymatic digestion method (~12 h). Finally, the purification of 2'-O-methylated ribonucleosides from RNA was successfully achieved in HeLa cells by the TiO2-based SPE method, which provided a proof-of-concept for the purification of relevant modified ribonucleosides from bulky normal ribonucleosides. Taken together, this strategy developed in the current study offers a promising option to purify 2'-deoxynucleosides/2'-O-modified ribonucleosides for their sensitive and accurate determination by eliminating normal ribonucleosides in biological samples.

Borated titania, a new option for the selective enrichment of cis-diol biomolecules.

As low abundance cis-diol biomolecules are of great significance in biological organisms, preparation of materials for the selective enrichment of such compounds is highly favorable for the development of the related proteomics and metabolomics. To this end, we have prepared monolithic borated titania by a non-aqueous sol-gel strategy as a new inorganic affinity material for the specific capture of nucleosides, glycopeptides and glycoproteins. Benefiting from the inorganic framework, this material prevented the hydrophobic interference, which was somewhat inevitable for the mainstream organic-based boronate affinity materials. The prepared material was carefully characterized by scanning electron microscope (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and nitrogen-sorption experiments to investigate the morphology and elemental composition. The excellent performance of borated titania on enrichment of cis-diol biomolecules was demonstrated by extracting the glycopeptides from horseradish peroxidase (HRP) digestion, standard glycoproteins, and nucleosides from a human-urine matrix. This kind of inorganic affinity material offers a new option for selective enrichment or separation of cis-diol biomolecules.

Determination of endogenous brassinosteroids in plant tissues using solid-phase extraction with double layered cartridge followed by high-performance liquid chromatography-tandem mass spectrometry.

INTRODUCTION: Brassinosteroids (BRs) are a group of important phytohormones that play vital roles in plant growth, development and a series of physiological phenomena. In order to understand biosynthesis, degradation and metabolic pathways of BRs, a reliable analytical method of BRs with effective sample pre-treatment process is favourable. OBJECTIVE: The development of a quick and effective method for the quantification of endogenous BRs in plant tissue with the aid of double layered solid-phase extraction (SPE) cartridges (graphite carbon black and primary secondary amine silica sorbent: GCB/PSA). METHOD: The method involved an initial extraction of BRs with acetonitrile, a dehydration process with anhydrous MgSO4 and NaCl, a SPE purification process with a double layered cartridge, and a further clean-up step utilising liquid-liquid extraction (LLE). The purification process was mainly realised on the GCB/PSA cartridge. GCB could eliminate hydrophobic compounds, especially those containing a π system, and PSA was introduced to remove the polar interferences. Endogenous BRs were quantified by HPLC-ESI-MS/MS. RESULTS: Good linearities were obtained in the range of 0.4-500 ng/mL (0.0124-15 ng), with the correlation coefficients above 0.9957. The relative recoveries of BRs of this method were in the range of 71.1-113.1%, with intra- and interday relative standard deviations (RSDs) less than 16.3%. With the proposed method, the requirement of plant tissue amount was minimised to 1 g fresh weight, which is the smallest amount reported so far, to our knowledge.

Evaluation of Vitamin D Status and the Analysis of Risk Factors of Vitamin D Deficiency in Twin Pregnancies.

OBJECTIVE: Optimization of maternal vitamin D (VD) status has beneficial effects on pregnancies, but little is known about it of twin pregnancies (TP). Our aim was to promote the current understanding of VD status and its associated factors in TP. METHODS: We performed liquid chromatography-tandem mass spectrometry to quantify 25-hydroxyvitamin D [25(OH)D] and used the enzyme-linked immunosorbent assay method to detect vitamin D binding protein (VDBP) in 218 singleton pregnancies (SP) and 236 TP. RESULTS: Levels of 25(OH)D and VDBP were higher in TP than SP. The 25(OH)D, free 25(OH)D, C-3 epimer of 25-hydroxyvitamin D [epi-25(OH)D], and VDBP all increased with gestational progress. Age, body mass index, and hemoglobin level were associated with VD deficiency (VDD). Analysis of covariance demonstrated that the 25(OH)D and VDBP of TP and SP still showed differences after adjusting for the above associated factors. CONCLUSION: Differences in VD status were found in SP and TP, suggesting that the assessment of VD status in TP should be treated with caution. High VDD prevalence is observed among pregnant Chinese women, and it is recommended to promote evaluation for VDD.

Rapid LC-MS/MS detection of 25-hydroxyvitamin D in dried blood spots.

BACKGROUND: The detection of 25-hydroxyvitamin D (25OHD) from dried blood spots (DBS) has been widely studied. However, the existing pretreatment methods suffer from limitations in terms of throughput (usually exceeding 2 h), complexity (involving liquid-liquid extraction or solid-phase extraction), and contamination (including multiple steps of organic solvent evaporation). RESULTS: We first released 25OHD from DBS samples by 50% acetonitrile solution through ultrasonication. Subsequently, the cold-induced phase separation technique was introduced for in-situ concentration and purification. Afterward, the PTAD derivatization of 25OHD was performed directly, profiting from the high acetonitrile content in the concentrated solution. In the end, the resulting solution was submitted to LC-MS/MS for quantification. The established LC-MS/MS methodology possessed favorable analytical performance, possessing lower limit of quantification of 1 ng/mL pointing to plasma, accuracy of 86.8-110.1% and imprecision of 5.4-16.8%. Method comparison with plasma samples demonstrated that over 93% of the detections met the acceptance limit for cross-validation of ±20%. SIGNIFICANCE AND NOVELTY: The novel sample preparation can be finished within 15 min and eliminated the traditional steps of extraction and organic solvent evaporation. Based on this high-throughput, reliable and applicable LC-MS/MS method, the detection of 25OHD in DBS samples can be better achieved for clinical patients and researchers with relevant demands.

Effects of COVID-19 infection in patients with autoimmune pulmonary alveolar proteinosis: a single-center study.

BACKGROUND: Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare interstitial lung disease. COVID-19 is associated with worse prognosis in previous lung diseases patients. But the prognosis of aPAP patients after infection with COVID-19 is unclear. In December 2022, China experienced a large-scale outbreak of Omicron variant of the SARS-CoV-2. In this study, we aim to explore the clinical outcomes of aPAP patients infected with COVID-19. RESULTS: A total of 39 aPAP patients were included in this study. 30.77% patients had a decrease in oxygen saturation after COVID-19 infection. We compared the two groups of patients with or without decreased oxygen saturation after COVID-19 infection and found that patients who had previous oxygen therapy (decreased oxygen saturation vs. non decreased oxygen saturation: 6/12 vs. 4/27, P = 0.043), with lower baseline arterial oxygen partial pressure (74.50 ± 13.61 mmHg vs. 86.49 ± 11.92 mmHg, P = 0.009), lower baseline DLCO/VA% [77.0 (74.3, 93.6) % vs. 89.5 (78.2, 97.4) %, P = 0.036], shorter baseline 6MWD [464 (406, 538) m vs. 532 (470, 575) m, P = 0.028], higher disease severity score (P = 0.017), were more likely to have decreased oxygen saturation after COVID-19 infection. CONCLUSION: aPAP patients with poor baseline respiration have a higher probability of hypoxia after COVID-19 infection, but fatal events were rare.

Determinants of Progression and Mortality in Lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis is a progressive diffuse cystic lung disease with approximately 85% survival at 10 years. The determinants of disease progression and mortality after the introduction of sirolimus therapy and vascular endothelial growth factor D (VEGF-D) as a biomarker have not been well defined. RESEARCH QUESTION: Which factors, including VEGF-D and sirolimus therapy, influence disease progression and survival prognosis in patients with lymphangioleiomyomatosis? STUDY DESIGN AND METHODS: The progression dataset and the survival dataset included 282 and 574 patients, respectively, from Peking Union Medical College Hospital, Beijing, China. A mixed-effects model was used to compute the rate of decline in FEV1, and generalized linear models were used to identify variables affecting FEV1 decline. A Cox proportional hazards model was used to explore the association between clinical variables and the outcomes of death or lung transplantation in patients with lymphangioleiomyomatosis. RESULTS: VEGF-D levels and sirolimus treatment were associated with FEV1 changes and survival prognosis. Compared with patients with VEGF-D of < 800 pg/mL at baseline, patients with VEGF-D of ≥ 800 pg/mL lost FEV1 faster (SE, -38.86 mL/y; 95% CI, -73.90 to -3.82 mL/y; P = .031). The 8-year cumulative survival rates of patients with VEGF-D of ≥ 2,000 pg/mL and < 2,000 pg/mL were 82.9% and 95.1%, respectively (P = .014). The generalized linear regression model also demonstrated the benefit of delaying the decline of FEV1 by 65.56 mL/y (95% CI, 29.06-102.06 mL/y) in patients treated with sirolimus compared with those without sirolimus (P < .001). The 8-year risk of death was reduced by 85.1% (hazard ratio, 0.149; 95% CI, 0.075-0.299) after sirolimus treatment. After inverse treatment probability weighting, the risks of death in the sirolimus group were reduced by 85.6%. CT scan results of grade III severity were associated with worse progression than results of grades I or II severity. Patients with baseline FEV1 of 70% predicted or St. George's Respiratory Questionnaire Symptoms domain 50 or higher predicted a higher risk of worse survival. INTERPRETATION: Serum VEGF-D levels, a biomarker of lymphangioleiomyomatosis, are associated with disease progression and survival. Sirolimus therapy is associated with slower disease progression and better survival in patients with lymphangioleiomyomatosis. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03193892; URL: www. CLINICALTRIALS: gov.

Facile preparation of SiO2/TiO2 composite monolithic capillary column and its application in enrichment of phosphopeptides.

A novel SiO(2)/TiO(2) composite monolithic capillary column was prepared by sol-gel technology and successfully applied to enrich phosphopeptides as a metal oxide affinity chromatography (MOAC) material. For the monolith preparation, tetramethoxysilane (TMOS) and tetrabutoxytitanium (TBOT) were used as silica and titania source, respectively, and glycerol was introduced to attenuate the activity of titanium precursor, which provided a mild synthetic condition. The prepared monolith was characterized by energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The results revealed an approximate 1/2 molar ratio of titanium to silica as well as an atom-scale homogeneity in the framework. The scanning electron microscopy (SEM) results demonstrated an excellent anchorage between the column and the inner capillary wall, and nitrogen adsorption-desorption experiments showed a bimodal porosity with a narrow mesopore distribution around 3.6 nm. The prepared monolith was then applied for selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA) as well as human blood serum, nonfat milk, and egg white using an in-tube solid phase microextraction (SPME) system. Our results showed that SiO(2)/TiO(2) composite monolithic capillary column could efficiently enrich the phosphopeptides from complex matrixes. To the best of our knowledge, this is the first attempt for preparing the silica-metal composite monolithic capillary column, which offers the promising application of the monolith on phosphoproteomics study.