超高效液相色谱-三重四极杆质谱联用方法测定血浆和尿液中的α-龙葵碱、α-卡茄碱和茄啶

建立了超高效液相色谱-三重四极杆质谱联用方法,检测血浆和尿液中的α-龙葵碱、α-卡茄碱和茄啶。样品经2%(v/v,下同)甲酸水溶液等量稀释,再经混合型阳离子交换固相萃取柱(MCX SPE)净化,以0.1%甲酸乙腈溶液和含0.05%甲酸的5 mmol/L乙酸铵水溶液作为流动相进行梯度洗脱,在UPLC BEH C18色谱柱上实现分离,正离子电喷雾串联质谱多反应监测(ESI-MS/MS MRM)方式检测,基质匹配外标法定量。一次进样分析时间为5.5min。血浆和尿液中3种待测物的线性范围均为0.3~100 ng/mL,相关系数为0.997~0.999;样品的检出限为0.1 ng/mL,定量限为0.3 ng/mL;血浆和尿液中的平均加标回收率分别为82%~112%和96%~114%,相对标准偏差为4.0%~16%和2.7%~17%(n=6)。方法简单、准确、灵敏,适用于马铃薯中毒检测。     

固相萃取-高效液相色谱/串联质谱法测定大鼠血浆中二十二碳六烯酸

建立了固相萃取-高效液相色谱/串联质谱(SPE-HPLC-MS/MS)测定大鼠血浆中二十二碳六烯酸(DHA)的分析方法。血浆样品经C18固相萃取(SPE)小柱净化后,采用Thermo C18色谱柱分离,以0.2%甲酸水溶液和乙腈为流动相,等度洗脱,在电喷雾离子源负离子模式下,采用质谱选择反应监测(SRM)模式检测,外标法定量。结果表明:DHA在0.10~60.0μg/mL范围内具有良好的线性关系(r2=0.9990);检出限(S/N=3)为0.04μg/mL,定量限(S/N=10)为0.10μg/mL;在2、10、30μg/mL 3个添加水平下,其平均回收率为94.0%~106.9%,方法的相对标准偏差(RSD)在2.15%~3.12%之间。该方法简单、快速,准确度、灵敏度高,适用于大鼠血浆中DHA的分析检测。     

液相色谱-串联质谱法测定人血浆中儿茶酚胺的含量

建立人血浆中儿茶酚胺液相色谱-串联质谱(LC-MS/MS)的检测方法。采用Agilent Zorbax Eclipse XDB-C18(4.6 mm×150 mm,5μm)柱子,柱温25℃,流动相为V(乙腈)∶V(0.1%甲酸溶液)=10∶90,流速0.6 mL/min,进样量20μL;电喷雾离子源(ESI),正离子检测,采用多反应监测(MRM)方式进行定量分析。肾上腺素、去甲肾上腺素和多巴胺的最小检测限(LOD)在4.3,2.4和6.6ng/mL之间,浓度在0~500 ng/mL范围内线性良好(r=0.9921),平均回收率在85.8%~93.4%之间,日内、日间精密度RSD为9%(n=5)。结果表明该方法专属性强、灵敏及准确,可以适用于人血浆儿茶酚胺的快速分析。     

全自动固相萃取-高效液相色谱-串联质谱法测定食糖中的雌二醇

建立了全自动固相萃取-高效液相色谱-串联质谱检测食糖中雌二醇含量的方法。食糖样品经水溶解后,用碱化乙腈提取,经HLB固相萃取柱净化,采用高效液相色谱-串联质谱分析,内标法定量。质谱分析采用电喷雾电离(ESI)负离子扫描,多反应监测(MRM)模式。雌二醇定量离子对为271.1/144.8和271.1/183.2,内标D2-雌二醇定量离子对为273.2/147.0。实验表明,经SPE柱净化后基质效应明显降低,样品中添加0.5~10 μg/kg的雌二醇,其回收率为83.9%~95.1%,相对标准偏差(n=6)小于10%。雌二醇的检出限为0.1 μg/kg,定量限为0.5 μg/kg。该方法提取效果好,具有良好的灵敏度、回收率和重复性。     

高效液相色谱-串联质谱法检测鸡肉中金霉素、差向金霉素

建立了高效液相色谱串联质谱(HPLC-MS/MS)快速测定鸡肉中差向金霉素、金霉素的检测方法.均质样品用pH4.0 EDTA-McIIvaine缓冲溶液提取,经Oasis HLB SPE小柱净化,采用LC-ESI-MS/MS和正离子多反应监测(MRM)模式检测.差向金霉素在0.025～0.8 μg/mL,金霉素在0.0...     

亚胺连接的多孔共价有机骨架材料结合固相萃取-液相色谱-串联质谱检测蜂蜜中雌激素

以亚胺连接的多孔共价有机骨架材料(IL-COF-1)作为固相萃取的吸附剂,建立了液相色谱-串联质谱快速检测蜂蜜样品中痕量雌激素的方法。该研究选择雌二醇、己烯雌酚、雌三醇、β-雌二醇和炔雌醇5种雌激素作为目标分析物。在蜂蜜样品中添加雌激素,采用单因素优化法对影响萃取效果的重要因素进行优化,获得最佳条件:IL-COF-1用量为30 mg,样品流速为3 mL/min,样品溶液pH值为7,以5 mL的1%(v/v)氨水-甲醇溶液进行洗脱,流速为0.4 mL/min,萃取过程中不添加NaCl。采用高效液相色谱-三重四极杆质谱联用技术对提取物中的雌激素进行定量分析。以乙腈和5 mmol/L的乙酸铵溶液作为流动相进行梯度洗脱,经C18色谱柱分离,采用电喷雾离子源、质谱多反应监测和负离子扫描模式,实现了蜂蜜样品中5种雌激素的快速定性定量分析。在最佳条件下,方法验证结果中雌三醇、β-雌二醇和炔雌醇的线性范围为1~500 ng/g,雌二醇和己烯雌酚的线性范围为0.1~100 ng/g,相关系数(r)为0.9934~0.9972。检出限(S/N=3)为0.01~0.30 ng/g,定量限(S/N=10)为0.05~0.95 ng/g。添加50 ng/g 5种雌激素进行重复性实验,日内精密度相对标准偏差(RSD)为3.2%~6.6%,日间精密度RSD为4.2%~7.9%。基于IL-COF-1的固相萃取-液相色谱-串联质谱法具有快速准确、灵敏度高等特点,适用于蜂蜜中雌激素的分析和检测。将该方法应用于4个实际蜂蜜样品中雌激素的检测,均未检出目标物;在低中高3个水平下,5种雌激素的加标回收率为80.1%~115.2%,结果令人满意。     

液相色谱-串联质谱法同时检测乙醛-DNA加合物

本文建立了一种同时检测DNA样品中Ethylidene-dG和Propano-dG含量的液相色谱-串联质谱(LC-MS/MS)方法,方法的精密度较好(RSD6%),检出限分别为0.010ng/mL和0.005ng/mL,回收率在97.2%~101.6%之间。同时,以体外小牛胸腺DNA为模型,选取不同乙醛暴露剂量(0、0.001、0.01、0.05、0.1、0.5和1.0mmol/L)和不同的暴露时间(0、2、4、10、12、20和24h),结果显示在体外Propano-dG加合物的生成需有氨基酸作为催化剂,且小牛胸腺DNA中乙醛-DNA加合物的含量随着染毒剂量和染毒时间的增加而升高,存在剂量-效应和时间-效应关系。     

UPLC-MS/MS法测定7类化妆品中4-氨基联苯

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)检测化妆品中4-氨基联苯的方法。化妆品样品经溶剂超声提取、净化(液液萃取或固相萃取),氮气吹至近干并定容后,通过高效液相色谱-串联四极杆质谱仪检测。分离柱为Waters Acquity BEH C18柱(1.7μm,2.1 mm×100 mm);流动相为0.3%甲酸水溶液-乙腈;流速0.5 mL·min-1。7种不同基质样品中4-氨基联苯的检测限均小于1.0ng·g-1。7种不同基质样品中4-氨基联苯的回收率为85.3%～111.2%,相对标准偏差(RSD)为0.93%～4.11%(n=3),在2.5ng·mL-1～250ng·mL-1浓度范围内呈良好的线性关系,线性回归系数r2均大于0.999。     

超高效液相色谱-串联质谱法测定辣椒制品中酸性橙Ⅱ

建立了辣椒制品中酸性橙Ⅱ的超高效液相色谱-串联质谱分析方法。样品用乙腈提取,WAX弱阴离子固相萃取柱净化浓缩,经超高效液相色谱分离,三重四极杆质谱电喷雾电离(ESI),负离子多反应监测模式检测。结果表明,在1~200ng/mL质量浓度范围内,线性相关系数大于0.998;加标回收率为83.2%~113.2%,相对标准偏差为0.7%~7.2%。检出限分别为1.0μg/kg(辣椒油及辣椒酱)和2.0μg/kg(辣椒粉)。方法适用于辣椒制品中酸性橙Ⅱ的测定。     

液相色谱-串联四极杆飞行时间质谱和超高效液相色谱-串联三重四极杆质谱用于检测牛肉中的刚果红

建立了牛肉中刚果红的检测方法。定性方法采用液相色谱-串联四极杆飞行时间质谱对未知物进行质谱谱图库匹配,定量分析采用超高效液相色谱-串联三重四极杆质谱。牛肉样品中的刚果红经液液萃取净化后,采用Agilent ZORBAX Eclipse Plus C₁₈ Rapid Resolution HD色谱柱(50 mm×2.1 mm, 1.8 μm)进行分离,流动相为95%(体积分数)甲醇,流速为0.2 mL/min。AB 4000⁺三重四极杆质谱仪在电喷雾负离子化(ESI)及MRM模式下定量。结果显示,刚果红在0.03~1 mg/L浓度范围内,线性关系良好(相关系数为0.9998),精密度良好(RSD小于5%),回收率为88%~91%,检出限约为0.01 mg/L。本方法快速简便,重现性好,可以为牛肉及其他肉制品中刚果红的定量提供良好的解决方案。     

Determination of ng/mL levetiracetam using ultra-high-performance liquid chromatography-photodiode absorbance

This paper demonstrates the analysis of levetiracetam, a new chiral antiepileptic drug, at ng/mL levels using an ultra-high-performance liquid chromatography (UHPLC)-photodiode absorbance (PDA) method. Three different sample preparation methods, liquid-liquid extraction with Extrelut, solid phase extraction (SPE) with Oasis HLB and Oasis MAX SPE cartridges, and protein precipitation with organic solvents were carried out. The last preparatory method is the simplest and provides the best recoveries: between 97.1% and 100.4% with RSD value below 5%. The column for separation is BEH C18 column (1.7 μm particle size and 100 × 2.1 mm i.d.) and acetonitrile-phosphate buffer (pH = 6.6; 0.01 M) (10/90 v/v) is the mobile phase. The results obtained are compared to analysis conducted by the HPLC method. The UHPLC method was validated in the range of 2-100 μg/mL levetiracetam concentration (R(2) = 0.9997). LOD and LOQ are 10 ng/mL and 33 ng/mL, respectively. The developed UHPLC method was applied to plasma samples of patient with epilepsy.     

Quantitative determination of piritramide in human plasma and urine by off- and on-line solid-phase extraction liquid chromatography coupled to tandem mass spectrometry

A method for the liquid chromatography/tandem mass spectrometric (LC/MS/MS) quantification of piritramide, a synthetic opioid, in plasma after conventional off-line solid-phase extraction (SPE) and in urine by on-line SPE-LC/MS/MS in positive electrospray mode was developed and validated. Applicability of the on-line approach for plasma samples was also tested. Deuterated piritramide served as internal standard. For the off-line SPE plasma method mixed cation-exchange SPE cartridges and a 150 x 2 mm C18 column with isocratic elution were used. For the on-line SPE method, a Waters Oasis HLB extraction column and the same C18 analytical column in a column-switching set-up with gradient elution were utilized. All assays were linear within a range of 0.5-100 ng/mL with a limit of detection of 0.05 ng/mL. The intra- and interday coefficients of variance ranged from 1.3 to 6.1% for plasma and 0.5 to 6.4% for urine, respectively. The extraction recovery for the off-line plasma assay was between 90.7 and 100.0%. Influence of matrix effects, and freeze/thaw and long-term stability were validated for both approaches; influence of urine pH additionally for quantification in urine.     

On-line solid phase extraction using the Prospekt-2 coupled with a liquid chromatography/tandem mass spectrometer for the determination of dextromethorphan, dextrorphan and guaifenesin in human plasma

An on-line liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure, using the Prospekt- 2 system, was developed and used for the determination of the levels of the active ingredients of cough/cold medications in human plasma matrix. The experimental configuration allows direct plasma injection by performing on- line solid phase extraction (SPE) on small cartridge columns prior to elution of the analyte(s) onto the analytical column and subsequent MS/MS detection. The quantitative analysis of three analytes with differing polarities, dextromethorphan (DEX), dextrorphan (DET) and guaifenesin (GG) in human plasma presented a significant challenge. Using stable-isotope-labeled internal standards for each analyte, the Prospekt-2 on-line methodology was evaluated for sensitivity, suppression, accuracy, precision, linearity, analyst time, analysis time, cost, carryover and ease of use. The lower limit of quantitation for the on-line SPE procedure for DEX, DET and GG was 0.05, 0.05 and 5.0 ng mL(-1), respectively, using a 0.1 mL sample volume. The linear range for DEX and DET was 0.05-50 ng mL(-1) and was 5-5,000 ng mL(-1) for GG. Accuracy and precision data for five different levels of QC samples were collected over three separate days. Accuracy ranged from 90% to 112% for all three analytes, while the precision, as measured by the %RSD, ranged from 1.5% to 16.0%     

Simultaneous determination of tryptophan,kynurenine, kynurenic acid,xanthurenic acid and 5‐hydroxytryptamine in human plasma by LC‐MS/MS and its application to acute myocardial infarction monitoring

Reliable methods for the determination of tryptophan and its metabolites are vital to the monitoring of biochemical states during the initiation and progression of cardiovascular disease. In the present study, a single‐run liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of tryptophan (Trp) and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), xanthurenic acid (XA) and 5‐hydroxytryptamine (5‐HT), in human plasma. The plasma samples were prepared using a protein precipitation approach, and the chromatographic separation was performed by gradient elution on a C₁₈ column within a total analysis time of 3.5 min. The calibration ranges were 40–20,000 ng/mL for Trp, 4–2000 ng/mL for Kyn, 0.2–100 ng/mL for KA, 0.4–200 ng/mL for XA and 1–500 ng/mL for 5‐HT, and the precision and accuracy were acceptable. The evaluation of recovery and internal standard‐normalized matrix effect proved that the sample preparation approach was effective and the matrix effect could be negligible. The newly developed method was successfully applied to the analysis of plasma samples from healthy individuals and myocardial infarction patients. The findings suggested that the plasma concentrations of Trp, Kyn, 5‐HT as well as the concentration ratios of Kyn/Trp and Trp/5‐HT might serve as biomarkers for the monitoring of acute myocardial infarction.     

On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples. II. Determination of clenbuterol in urine using multiple-stage mass spectrometry in an ion-trap mass spectrometer

Solid-phase extraction (SPE) was coupled to ion-trap mass spectrometry to determine clenbuterol in urine. For SPE a cartridge exchanger was used and, after extraction, the eluate was directly introduced into the mass spectrometer. For two types of cartridges, i.e. C18 and polydivinylbenzene (PDVB), the total SPE procedure (including injection of 1 mL urine, washing, and desorption) has been optimised. The total analysis, including SPE, elution, and detection, took 8.5 min with PDVB cartridges, while an analysis time of 11.5 min was obtained with C18 cartridges. A considerable amount of matrix was present after extraction of urine over C18 cartridges, resulting in significant ion suppression. With PDVB cartridges, the matrix was less prominent, and less ion suppression was observed. For single MS, a detection limit (LOD) of about 25 ng/mL was found with PDVB cartridges. With C18 cartridges an LOD of only about 50 ng/mL could be obtained. Applying tandem mass spectrometry (MS/MS) did not lead to an improved LOD due to an interfering compound. However, a considerable improvement in the LOD was obtained with MS3. The selectivity and sensitivity were increased by the combination of efficient fragmentation of clenbuterol and reduction of the noise. Detection limits of 2 and 0.5 ng/mL were obtained with C18 and PDVB cartridges, respectively. The ion suppression was 4 to 45% (concentration range: 250 to 1.0 ng/mL) after extraction of urine using PDVB cartridges, and up to 70% ion suppression was observed using C18 cartridges. With MS4, no further improvement in selectivity and sensitivity was achieved, due to inefficient fragmentation of clenbuterol and no further reduction of noise.     

A new approach to the effective preparation of plasma samples for rapid drug quantitation using on-line solid phase extraction mass spectrometry.

A procedure that permits rapid development of an optimized solid phase extraction (SPE) method for the analysis of drugs in plasma by on-line solid phase extraction-mass spectrometry (SPE-MS) has been developed. This procedure employs the concept of manipulating the pH and the percentage of organic solvent in the chromatographic mobile phase to affect the retention behaviors of both the matrix components and the analytes of interest. This resulted in the effective removal of matrix interferences from biological samples during SPE. During a the method development, only generic HPLC gradient approaches were needed, and multiple samples were pooled so that several SPE methods could be investigated at once. The analysis time per sample was 1.3 minutes. Thus, the time involved in the entire method development (analysis of a set of samples) was less than one hour. With the knowledge of the retention behaviors of the analytes with respect to the pH and the percentage of organic, it was then possible to compose an optimized SPE-MS method. This method consisted of a base/organic and then an acid/organic washing step, followed by a rapid gradient elution step. Due to the rigorous washing procedure, most matrix interferences were removed, and analytes eluted off the SPE sorbent suffered from very little matrix interference. Thus, quantitation of drugs in plasma by a single quadrupole mass spectrometer could be accomplished, something that was not possible when only a generic gradient was used for on-line SPE-MS. In addition, both external and internal calibration curves could be obtained for the concentration range from 5 to 500 ng/mL with correlation coefficients of 0.99 (using 1/x as a weighting factor) and relative standard deviations (RSDs) less than 10%. The results achieved were comparable to those obtained by the use of a triple quadrupole mass spectrometer. Moreover, the robustness of the method was tested by continuously injecting plasma samples. During 136 runs, the absolute peak area variation for these three basic drugs was less than 15% without taking the signal variation from the mass spectrometer into account. Significantly, the on-line developed method can be directly transferred to a 96-well format SPE plate.     

芥子气中毒人员尿液标记物谱分析及其临床诊断应用

该研究基于暴露生物标记物溯源思路,应用于解决芥子气(SM)临床早期诊断、溯源确证难题。建立了芥子气中毒患者尿液中7种游离代谢产物的两步固相萃取/超高效液相色谱-串联质谱(SPE/UPLC-MS/MS)同时定量方法,检出限为5 pg/mL~1 ng/mL,定量下限为10pg/mL~5 ng/mL;结合前期建立的4种游离碱基加合物的同位素稀释-UPLC-MS/MS定量方法,对1例疑似芥子气中毒人员尿液中可能赋存的生物标记物进行了全筛查分析。尿液中共检出3类10种生物标记物,包括首次报道的游离代谢产物芥子亚砜,可确证患者为芥子气中毒;除硫二甘醇外,标记物含量均在暴露后3~4 d达到峰值,随后降低,至7 d仍可检出,其中谷胱甘肽加合物的β裂解产物含量相对较高,可作为芥子气中毒早期诊断与疗效评估的重要指标。     

Pharmacokinetic and bioavailability study of kurarinone in dog plasma by UHPLC–MS/MS

Kurarinone, a natural prenylated flavonone isolated from Sophora flavescens, has been exhibited various activities. This study aimed to establish a simple and sensitive ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for determining kurarinone in dog plasma. Acetonitrile-mediated precipitation was applied for sample pretreatment. Chromatographic separation was achieved on a Waters ACQUITY HSS T3 (100 × 2.1 mm, i. d., 1.8 μm) column with gradient elution using water containing 0.1% formic acid and acetonitrile as mobile phase. Quantitation was performed using an electrospray ionization source in negative multiple reaction monitoring mode. The linearity of this method was over the concentration range 0.1–500 ng/mL with the lowest limit of quantification (LLOQ) of 0.1 ng/mL. The intra- and inter-day precision was less than 10.51% and the accuracy ranged from 94.85% to 97.72%, respectively. The extraction recovery of kurarinone in dog plasma was more than 82.37% and no significant matrix effect was observed. The analyte was stable under tested storage conditions. The validated method was further successfully applied to a preclinical pharmacokinetic study of kurarinone in dog after a single intravenous (2 mg/kg) and oral (20 mg/kg) administration. The results revealed that kurarinone was rapidly absorbed into plasma with good bioavailability (38.19%) and low clearance.     

Determination of Ginsenoside Rg2 in Rat Plasma by High‐Performance Liquid Chromatography–Mass Spectrometry after Solid‐Phase Extraction

Abstract

A sensitive liquid chromatography‐mass spectrometric (LC/MS) method for the quantification of ginsenoside Rg2 (Rg2) in rat plasma was developed after solid‐phase extraction (SPE). Chromatographic separation was achieved on a reversed‐phase Kromasil C₁₈ column with the mobile phase of acetonitrile‐ammonium chloride (500 µM/L) and step gradient elution resulted in a total run time of about 9 min. The analytes were detected using electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range (5–2500 ng/mL) (r=0.9999). Limit of quantification (LOQ) was 5 ng/mL and the limit of detection (LOD) was 2 ng/mL using 100 µL plasma sample. Average recoveries ranged from 72.43–84.73% in plasma at the concentrations of 20, 200, and 2000 ng/mL. Intra‐ and interday coefficients of variation for the assay were 4.93–10.87% and 4.06–7.84%, respectively. The method was successfully applied to the analysis of ginsenoside Rg2 in rat plasma. The applicability of this assay was examined in a preliminary pharmacokinetic study of ginsenoside Rg2 in rats.     

Simultaneous determination of aurantio‐obtusin,chrysoobtusin, obtusin and 1‐desmethylobtusin in rat plasma by UHPLC‐MS/MS

A sensitive and reliable ultra‐high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC‐MS/MS) method was developed and validated for the simultaneous determination of four active components of Semen Cassiae extract (aurantio‐obtusin, chrysoobtusin, obtusin and 1‐desmethylobtusin) in rat plasma after oral administration. Chromatographic separation was achieved on an Agilent Poroshell 120 C₁₈ column with gradient elution using a mobile phase that consisted of acetonitrile‐ammonium acetate in water (30 mm ) at a flow rate of 0.4 mL/min. Detection was performed by a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The calibration curve was linear over a range of 3.24–1296 ng/mL for aurantio‐obtusin, 0.77–618 ng/mL for chrysoobtusin, 34.55–1818 ng/mL for obtusin and 1.86–1485 ng/mL for 1‐desmethylobtusin. Inter‐ and intra‐day assay variation was <15%. All analytes were shown to be stable during all sample storage and analysis procedures. Copyright © 2013 John Wiley & Sons, Ltd.