原位磁固相萃取/液相色谱-串联质谱检测人血清中25-羟基维生素D北大核心CSCD

本研究提出一种原位磁固相萃取法并联合液相色谱-串联质谱(LC-MS/MS)检测人血清中25-羟基维生素D 2[25(OH)VD 2]和25-羟基维生素D 3[25(OH)VD 3]。研究利用原位磁固相萃取法高效提取人血清样本中25(OH)VD,联合LC-MS/MS测定人血清中25(OH)VD 2和25(OH)VD 3,并考察了方法的线性范围、定量下限、基质效应、精密度与准确度。实验结果表明,原位磁固相萃取法联合LC-MS/MS检测人血清25(OH)VD 2和25(OH)VD 3的检测限均为0.5 ng/mL,线性范围均为1~100 ng/mL,回收率范围为98%~103%,日内精密度与日间精密度均在7%以内。因此,原位磁固相萃取法可有效净化复杂生物样本,联合LC-MS/MS可便捷、快速、准确检测临床上人血清样本中25(OH)VD,有望应用于更多复杂样本基质下痕量化合物的检测场景。     

食品中维生素D与25-羟基维生素D检测技术及含量分布研究进展

维生素D是一种对于维持人体健康具有重要作用的脂溶性维生素,25-羟基维生素D是其在人体内循环和存储的主要形式。食品中维生素D和25-羟基维生素D前处理的通常采用碱皂化、有机溶剂提取、固相萃取或者半制备色谱净化;其测定方法多为放射免疫法和液相色谱法。液相色谱串联质谱凭借高灵敏度和高准确度,目前在食品中维生素D和25-羟基维生素D测定中发挥重要作用。近年来二维液相色谱和超高效超临界流体色谱由于其强大的分离能力,在食品中维生素D和25-羟基维生素D的分析中表现出强大的潜力。该文综述了近年来食品中维生素D和25-羟基维生素D的检测方法及二者在动物食品和植物食品中的含量分布研究,以期为建立适合不同食物样品的测定方法,指导居民合理膳食,进行膳食摄入量评估等研究工作提供参考。     

Vitamin D3在儿童桥本甲状腺炎的水平及对IL-4、IFN-γ的影响

通过体外实验,直接从T细胞水平,在1α,25(OH)2D3的干预下,分别以IFN-γ、IL-4作为Th1、Th2的代表,分析人周围血单个核细胞(PBMC)中IFN-γ和IL-4在HT中的细微变化以及血清25(OH)D3水平,探讨1α,25(OH)2D3对HT细胞因子的影响。以27例HT患儿为研究对象,17名健康儿童作对照,采集静脉血,部分用于分离其PBMC,并用PHA活化T细胞并培养,设1α,25(OH)2D3干预组和对照组,收集培养上清液。ELISA检测上清液IL-4、IFN-γ水平。同时另一部分静脉血分离出血清,并提取25(OH)D3,用放射免疫法(RIA)测定其水平。结果表明,HT组25(OH)D3水平显著低于健康对照,分别为(21.85±5.73)ng/mL和(27.56±7.46)ng/mL(P<0.01),且HT组PBMC产生IFN-γ水平显著高于健康对照,分别为(2 146.15±355.01)pg/mL和(1 462.00±101.52)pg/mL(P<0.01),而IL-4的表达显著低于健康对照,分别为(52.26±7.17)pg/mL和(59.32±4.21)pg/mL(P<0.01)。1α,25(OH)2D3干预后HT患儿IFN-γ表达显著下调,为(1 536.00±243.95)pg/mL(P<0.01),IL-4的变化不明显。提示HT患儿的25(OH)D3水平严重不足,1α,25(OH)2D3能通过抑制HT增强的IFN-γ分泌,纠正Th1、Th2细胞因子失衡,理论上1,α25(OH)2D3可改善儿童HT Th1、Th2细胞因子失衡。     

Vitamin D3对儿童桥本甲状腺炎IL-10、IL-12的调节作用

目的:分别以IL-12、IL-10作为Th1、Th2的代表,在1,α25(OH)2D3的干预下,在单核细胞水平分析人外周血单核细胞(PBMC)中IL-10、IL-12在儿童桥本甲状腺炎(HT)中的细微变化,并探讨1α,25(OH)2D3的免疫调节作用。方法:以27例HT患儿为研究对象,17名健康儿童作对照,采集外周静脉血,一部分用于分离并培养单核细胞,设1,α25(OH)2D3干预组和对照组,收集培养上清液,ELISA检测上清液中IL-10、IL-12水平。另一部分静脉血分离出血清,再提取25(OH)D3,用放射免疫法(RIA)测定25(OH)D3水平。结果:HT组25(OH)D3显著低于健康对照,分别为(21.85±5.73)ng/mL和(27.56±7.46)ng/mL(P<0.01),且HT组PBMC产生IL-12水平显著高于健康对照,分别为(119.18±28.65)pg/mL和(102.84±23.86)pg/mL(P<0.01),而IL-10的表达显著低于健康对照,分别为(132.99±12.04)pg/mL和(171.41±35.72)pg/mL(P<0.01)。1α,25(OH)2D3干预后HT患儿IL-12表达显著下调为(98.57±11.98)pg/mL(P<0.01),IL-10表达在HT组和健康对照组均上调,分别为(184.15±35.34)pg/mL(P<0.01)、(223.77±53.36)pg/mL(P<0.01),HT组ρ(IL-10)/ρ(IL-12)比值显著升高。结论:HT患儿的25(OH)D3水平不足。1α,25(OH)2D3参与单核细胞分泌细胞因子的调节过程,能抑制HT增强的Th1型细胞因子IL-12分泌,增加IL-10表达,纠正n(Th1)/n(Th2)细胞因子失衡。     

活性维生素D3类似物的合成及构效关系研究

1α,25-二羟基维生素D₃(1α,25-(OH)₂-D₃,125D)是维生素D中最具生理活性的代谢产物,但因高钙血症副反应而限制其临床应用。从对构效关系的研究出发,迄今已合成三千多种类似物。本文综述了近年来对某些A环修饰(C2位修饰、C3位修饰、芳香A环类似物、A环开环类似物)、侧链修饰、CD环修饰、seco-B环修饰和非开环甾体的活性维生素 D₃ 类似物的设计、合成以及构效关系的研究,旨在为新型较佳活性维生素D₃类似物的合成及临床开发提供参考。     

HPLC同时测定保健食品中维生素A、D3、E和β-胡萝卜素

建立了一种同时测定保健食品中维生素A、D3、E、β-胡萝卜素的高效液相色谱法。样品皂化后乙醚提取,旋转蒸干,流动相溶解,HPLC紫外检测。实验结果显示,VA、VD3、VE和β-胡萝卜素的线性范围分别为0.012~500、0.030~500、0.12~500和0.015~500μg/mL,且浓度范围内线性良好;检出限分别为0.24、0.60、2.40和0.30μg/g;相对标准偏差分别为1.9%、3.4%、4.6%和2.9%;VA、VD、VE和β-胡萝卜素的加标回收率为84.8%~119.0%。分析周期在20 min以内,方法适合保健食品样品的分析。     

高效液相色谱-串联质谱法测定食品中维生素D_2和维生素D_3

提出了食品中维生素D2和维生素D3的高效液相色谱-串联质谱分析方法。食品样品经氢氧化钾皂化后乙醚萃取,所得有机相经无水硫酸钠除水后蒸发至干。用1 mL乙醇溶解后经Agilent Zorbax XDB C18色谱柱(2.1 mm×50 mm,3.5μm)分离,用甲-醇10 mmol·L-1乙酸铵溶液(90+10)的混合溶液洗脱,采用电喷雾正离子模式多反应监测。维生素D2和维生素D3的线性范围均为50~500μg·L-1,检出限(3S/N)均为10μg·L-1。方法应用于测定奶粉和酸酸乳中维生素D2和维生素D3,回收率在76.8%~83.9%之间。     

超临界色谱法同时测定化妆品中维生素A类衍生物和维生素D2,D3

采用超临界色谱法建立了同时测定化妆品中维生素A乙酸酯、维生素A丙酸酯、维生素A棕榈酸酯、维生素D2和D3的分析方法。水溶性化妆品和油溶性化妆品经不同比例的水-乙腈-正己烷溶剂体系按不同的添加顺序进行分散、提取后,采用Viridis BEH 2-EP色谱柱(250×4.6 mm,5μm),以CO2为流动相,异丙醇-正己烷(1∶1)为改性剂,进行梯度洗脱分离,光电二极管矩阵检测器(SFC-PDA)检测,结合保留时间和光谱图定性,外标标准曲线法定量。实验结果表明,5种目标物质量浓度在1.0~60 mg/L范围内线性关系良好,相关系数为0.9992~0.9996;维生素A乙酸酯、维生素A丙酸酯、维生素A棕榈酸酯的定量下限(S/N=10)为4.0 mg/kg,维生素D2和D3的定量下限(S/N=10)为8.0 mg/kg。空白基质加标回收率为93.8%~110.1%,相对标准偏差(RSDs)小于13%。方法适用于各类化妆品中维生素A乙酸酯、维生素A丙酸酯、维生素A棕榈酸酯、维生素D2和D3的测定。     

新试剂7-(2,4,6-三羟基苯偶氮)-8-羟基喹啉-5-磺酸光度法同时测定微量锆和铪

本文研究了新试剂7-(2,4,6-三羟基苯偶氮)-8-羟基喹啉-5-磺酸(THBAQS)与锆、铪的显色反应.酸性条件下锆、铪均可形成 M-THBAQS-CTMAB三元络合物,ε_(315nm)~(Zr)=3. 27×10~4,ε_(515nm)~(HI)=3.8×10~4,适量H_2O_2使锆络合物吸光度显著降低,据此实现了锆铪的同时测定.本法简便快速、适用范围为0～30μgZr/25 mL,0～25μg Hf/25 mL,且X_(Zr)+X_(Hf）<35μg/25 mL.     

13α-取代-去-C-环-19-去亚甲基-1α,25-二羟基维生素D_3类似物的合成

以维生素D3为起始原料,经臭氧化,Baeyer-Villiger氧化内酯化,开裂六元环,二氧化钌(RuO2)引进C-25位羟基,最后经Horner-Wittig反应偶合D环和A环得到13-取代-去-C-环-19-去亚甲基-1α,25-二羟基维生素D3类似物(12a,12b).     

Development,validation and comparison of four methods for quantifying endogenous 25OH‐D3 in human plasma

To meet the increasing clinical needs for 25‐hydroxyvitamin D3 (25OH‐D3) detection, the development of an efficient and accurate high‐performance liquid chromatography–mass spectrometry (HPLC–MS) method for plasma 25OH‐D3 quantitation is important. Since 25OH‐D3 is an endogenous compound, the lack of a plasma blank increases the difficulty of accurately quantifying 25OH‐D3. Selection of a method suitable for clinical monitoring among various methods for endogenous compound quantification is necessary. Methyl tert butyl ether was chosen for the sample treatment in a liquid–liquid extraction protocol. Water as a blank matrix, 5% human serum albumin in water as a blank matrix, surrogate analyte and background subtraction were designed to address the problem of a deficiency of a plasma blank. Four liquid chromatography–tandem mass spectrometry methods were fully validated to verify the advantages and limitations owing to regulatory deficiencies for endogenous compound validation. All four methods met the criteria and could be used to monitor clinical samples. Overall 30 human plasma samples were quantified in parallel using the four methods. The difference between any two methods was <12.6% and the total relative standard deviation was <5.2%. Background subtraction and 5% human serum albumin in water as a blank matrix may be better choices considering data quality, matrix similarity, cost and practicality.     

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25‐hydroxyvitamin D₃ [25(OH)D₃, the best‐established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI‐MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels–Alder reaction with 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione followed by acetylation, to enhance the detectability of 25(OH)D₃ in ESI‐MS/MS and to separate 25(OH)D₃ from 3‐epi‐25‐hydroxyvitamin D₃ [3‐epi‐25(OH)D₃], a potent interfering metabolite. 25(OH)D₃ was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 μL of whole blood), purified using an Oasis HLB^® cartridge, and subjected to derivatization prior to analysis with LC/ESI‐MS/MS. 25‐Hydroxyvitamin D₄ was used as the internal standard. This method was reproducible (intra‐ and inter‐assay RSDs, <6.9%) and accurate (analytical recovery, 95.2–102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D₃ in neonatal DBSs and detection of vitamin D deficiency without interference from 3‐epi‐25(OH)D₃.     

Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50 x 2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1alpha,25(OH)2D3 and the internal standard [2H6]1alpha,25(OH)2D3 were detected by using MS-MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml(-1) for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1alpha,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.     

液相色谱-串联质谱法检测干血点中的同型半胱氨酸

建立了一种高通量液相色谱-串联质谱技术检测干血点(DBS)中同型半胱氨酸(homocycteine, Hcy)的方法。以DBS为样本,homocystine-D8为同位素内标,二硫苏糖醇(DTT)为蛋白结合态Hcy的还原剂,使用含0.1%(v/v)甲酸、0.05%(v/v)三氟乙酸的乙腈溶液萃取。整个前处理过程使用自动移液平台及96孔板实现高通量自动化操作。处理后的样本经过Phenomenex CN柱分离,使用多反应监测模式进行LC-MS/MS分析。结果表明:Hcy的检出限为0.12 μ mol/L(S/N=3),定量限为0.46 μ mol/L(S/N=10)。Hcy在1.16~148.00 μ mol/L范围内线性关系良好,R²=0.994。Hcy的平均回收率为(103.0±4.97)%~(112.0±2.13)%,日内相对标准偏差(RSD)为1.9%~4.6%,日间RSD为1.5%~7.1%。DBS样本在不同温度(-4、-20、22和37℃)下储存不同时间(0、1、2、3、4、5、6、14天)后的稳定性试验显示样本总体RSD<15%,经前处理后的样本在48 h内的稳定性试验显示样本总体RSD<5%。该方法与传统生化分析方法的相关性好(R²=0.9818, n=47)。     

An LC-MS/MS Method for Analysis of Vitamin D Metabolites and C3 Epimers in Mice Serum: Oral Supplementation Compared to UV Irradiation

Introduction: The most common forms of vitamin D in human and mouse serum are vitamin D3 and vitamin D2 and their metabolites. The aim of this study is to determine whether diet and sunlight directly affect the circulating concentrations of vitamin D metabolites in a mouse model. We investigated the serum concentrations of eight vitamin D metabolites—vitamin D (vitamin D3 + vitamin D2), 25OHD (25OHD3 + 25OHD2), 1α25(OH)₂D (1α25(OH)₂D2, and 1α25(OH)₂D3)—including their epimer, 3-epi-25OHD (3-epi-25OHD3 and 3-epi-25OHD2), and a bile acid precursor 7alpha-hydroxy-4-cholesten-3-one (7αC4), which is known to cause interference in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Method: The LC-MS/MS method was validated according to FDA-US guidelines. The validated method was used for the analysis of mouse serum samples. Forty blood samples from mice were collected and divided into three groups. The first group, the DDD mice, were fed a vitamin D-deficient diet (25 IU VD3/kg of diet) and kept in the dark; the second group, the SDD mice, were maintained on a standard-vitamin D diet (1000 IU VD3) and kept in the dark; and the third group, SDL, were fed a standard-vitamin D diet (1000 IU VD3) but kept on a normal light/dark cycle. LC-MS/MS was used for the efficient separation and quantitation of all the analytes. Results: The validated method showed good linearity and specificity. The intraday and interday precision were both <16%, and the accuracy across the assay range was within 100 ± 15%. The recoveries ranged between 75 and 95%. The stability results showed that vitamin D metabolites are not very stable when exposed to continuous freeze–thaw cycles; the variations in concentrations of vitamin D metabolites ranged between 15 and 60%. The overlapping peaks of vitamin D, its epimers, and its isobar (7αC4) were resolved using chromatographic separation. There were significant differences in the concentrations of all metabolites of vitamin D between the DDD and SDL mice. Between the groups SDD (control) and SDL, a significant difference in the concentrations of 3-epi-25OHD was noted, where C3 epimer was about 30% higher in SDL group while no significant differences were noted in the concentrations of vitamin D, 25OHD, 1α25(OH)₂D, and 7αC4 between SDD and SDL group. Conclusions: A validated method, combined with a simple extraction technique, for the sensitive LC-MS/MS determination of vitamin D metabolites is described here. The method can eliminate the interferences in LC-MS/MS analysis caused by the overlapping epimer and isobar due to them having the same molecular weights as 25OHD. The validated method was applied to mouse serum samples. It was concluded that a standard-vitamin D diet causes an increase in the proportion of all the vitamin D metabolites and C3 epimers and isobar, while UV light has no pronounced effect on the concentrations of the majority of the vitamin D metabolites except 3-epi-25OHD. Further studies are required to confirm this observation in humans and to investigate the biochemical pathways related to vitamin D’s metabolites and their epimers.     

Synthesis of an optically active intermediate for A-ring of 19-nor- 1α,25-dihydroxyvitamin D3

An optically active intermediate 5 for A-ring of 19-nor-1a,25-dihydroxyvitamin D3 2 has been synthesized in five steps, starting from readily available, inexpensive D( )-xylose 6 with good yield.     

Comparative evaluation of new Cookson‐type reagents for LC/ESI‐MS/MS assay of 25‐hydroxyvitamin D3 in neonatal blood samples

The screening of vitamin D deficiency in neonatal infants, which is based on the blood 25‐hydroxyvitamin D₃ [25(OH)D₃] quantification, is important for the early detection, diagnosis and health risk assessment of several diseases. In this study, two new Cookson‐type reagents, 4‐(4‐diethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DEAPTAD) and 4‐(6‐quinolyl)‐1,2,4‐triazoline‐3,5‐dione, were designed and synthesized, then compared with the previous reagents, 4‐phenyl‐1,2,4‐triazoline‐3,5‐dione (PTAD) and 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD), in terms of sensitivity and specificity in the assay of 25(OH)D₃ in neonatal blood samples by liquid chromatography/electrospray ionization–tandem mass spectrometry. Among the reagents, DEAPTAD was found to be the most promising. The limit of detection (0.38 fmol on the column) of the DEAPTAD‐derivatized 25(OH)D₃ was 60 and 2 times lower than those of the intact 25(OH)D₃ and the PTAD derivative, respectively. 25(OH)D₃ was more clearly detected in the plasma sample as the DEAPTAD derivative than the DAPTAD derivative owing to the lower background noise. DEAPTAD derivatization was also useful for the separation of 25(OH)D₃ from a potent interfering metabolite, 3‐epi‐25‐hydroxyvitamin D₃. By using DEAPTAD, a trace amount of 25(OH)D₃ in dried blood spots was reproducibly determined without interference from coexisting compounds. Thus, DEAPTAD was proved useful in the measurement of 25(OH)D₃ in neonatal blood samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

A simple micro‐extraction plate assay for automated LC‐MS/MS analysis of human serum 25‐hydroxyvitamin D levels

This short application note describes a simple and automated assay for determination of 25‐hydroxyvitamin D (25(OH)D) levels in very small volumes of human serum. It utilizes commercial 96‐well micro‐extraction plates with commercial 25(OH)D isotope calibration and quality control kits. Separation was achieved using a pentafluorophenyl liquid chromatography column followed by multiple reaction monitoring‐based quantification on an electrospray triple quadrupole mass spectrometer. Emphasis was placed on providing a simple assay that can be rapidly established in non‐specialized laboratories within days, without the need for laborious and time consuming sample preparation steps, advanced calibration or data acquisition routines. The analytical figures of merit obtained from this assay compared well to established assays. To demonstrate the applicability, the assay was applied to analysis of serum samples from patients with chronic liver diseases and compared to results from a routine clinical immunoassay. Copyright © 2015 John Wiley & Sons, Ltd.     

Association of 25-hydroxyvitamin D3)levels in adult New Zealanders with ethnicity, skin color and self-reported skin sensitivity to sun exposure

The study aim was to determine the contribution of ethnicity, objectively measured skin color and skin reaction-to-sun exposure to variations in 25-hydroxyvitamin D(3) [25(OH)D(3) ]. A multiethnic sample (European, Maori, Pacific and Asian) of 503 adult volunteers aged 18-85 years, recruited from Auckland and Dunedin in New Zealand, answered a questionnaire on sun exposure and self-defined ethnicity. Skin color was measured using a spectrophotometer and the Individual Typology Angle (ITA) calculated. A blood sample was collected 4 weeks later to measure 25(OH)D(3). 25(OH)D(3) was associated with ethnicity, but not self-reported skin reaction-to-sun exposure. Amongst the ethnic groups, Asians had the lowest mean 25(OH)D level (37.0 nmol L(-1)) and Europeans with lighter colored skin had the highest (57.9 nmol L(-1)). An association also was seen between 25(OH)D(3) and skin color, with an increase of 2-3 nmol L(-1) per 10° increase in ITA value, indicating higher 25(OH)D(3) with lighter skin color; but much of this association disappeared after adjusting for ethnicity. In contrast, ethnicity remained associated with 25(OH)D(3) after adjusting for ITA skin color and skin reaction-to-sun exposure. These results indicate that self-defined ethnicity was a major determinant of variations in serum 25(OH)D(3), while objective measures of skin color explained relatively little additional variation.