液相色谱串联质谱法检测人血清雌酮、雌二醇

目的建立同时测定人血清中雌酮(E1)及雌二醇(E2)水平的液相色谱串联质谱(LC-MS/MS)法,并初步应用于临床血清样品的雌激素检测。方法血清样品经乙酸乙酯提取,丹磺酰氯衍生化后进行LC-MS/MS分析。用Agilent ZORBAX SBC18色谱柱进行线性梯度洗脱,流动相为乙腈(0.05%甲酸)-水(0.05%甲酸),流速为0.3 m L/min。用多重反应离子监测(MRM)正离子模式对血清中的E1、E2进行质谱检测,并根据"生物样品定量分析方法验证指导原则(中国药典,2015)"对该方法的特异性、线性范围、灵敏度、精密度、提取回收率和稳定性等进行评价。用本法对172例健康成年女性血清标本进行E1、E2检测,用百分位数法计算不同月经周期E1、E2的浓度区间。结果 LC-MS/MS法可同时检测人血清中E1、E2的含量,在0.05~10 nmol/L范围内线性关系良好。该法的定量限为0.05 nmol/L,日内与日间不精密度均小于15%,稳定性良好。初步临床应用显示,用LC-MS/MS法计算所得的E1、E2浓度区间符合女性月经周期生理性变化规律。结论 LC-MS/MS法能有效分离并定量检测人血清中E1、E2的浓度水平,其线性范围广、灵敏度高、精密度好,有望作为临床血清雌激素检测的参考方法。     

自动化学发光免疫分析仪检测血清25-羟基维生素D的性能评价

目的对自动化学发光免疫分析仪测定25-羟基维生素D的检测性能进行验证和评估,以确定是否满足临床检测的要求。方法根据美国国家临床实验室标准化委员会的EP5-A2方案评价精密度,EP17-A方案评价灵敏度,EP6-A2方案评估线性范围,并采用雅培全自动化学发光法和液相色谱串联质谱法(LC-MS/MS)分别测定50例健康体检儿童的血清25-羟基维生素D,比较测定结果的相关性和偏倚。结果雅培全自动化学发光法检测的精密度和灵敏度符合试剂盒的声明,可以接受。线性范围涵盖了健康者和绝大多数患者的检测水平,可以满足临床需要。自动化学发光免疫分析仪对50例体检儿童血清标本的检测结果与当前作为"金标准"的LC-MS/MS相比,结果符合性较好。结论自动化学发光免疫分析仪测定25-羟基维生素D在精密度、灵敏度、线性范围方面均符合要求,对临床血清标本的25-羟基维生素D的检测结果也与当前作为"金标准"的LC-MS/MS符合性较好,可以满足临床检测的要求。     

apo A1和apo B侯选参考方法在室间质量评价中的应用价值

目的 探讨载脂蛋白(apo)A1和apo B的候选参考方法 在室间质量评价(EQA)中的应用价值.方法 以5个水平的冰冻人血清制品作为EQA质评物,采用免疫透射比浊法(ITA)检测.采用同位素稀释液相色谱串联质谱(ID-LC-MS/MS)定量技术对EQA样本进行定值,包括变性、烷基化和胰蛋白酶酶解处理,使用C18色谱柱...     

液相色谱-串联质谱法监测血清茶碱浓度及室间质量评价

目的对液相色谱-串联质谱(LC-MS/MS)检测茶碱进行方法学评价,探讨其在茶碱治疗药物监测(TDM)中的应用。方法在血清添加放射性核素内标茶碱-D6,经蛋白沉淀稀释后采用LC-MS/MS测定。以Capcell C18 MGⅢ(100 mm×2.0 mm,5μm)为分析柱进行反相色谱分离;以0.1%甲酸乙腈-0.1%甲酸水[20∶80(v/v)]为流动相,流速为0.3 m L/min;以电喷雾离子化串联四级杆质谱、正离子多反应监测进行定量检测。用建立的方法从2008年起连续参加卫生部临检中心茶碱TDM室间质量评价。结果 LC-MS/MS检测茶碱的线性范围为1~50μg/m L,批内和批间精密度分别为2.26%~6.65%和4.70%~6.84%,准确度分别为94.14%~104.00%。单个样品的监测分析时间为3.5 min。冻融(-30℃室温反复解冻3次)、室温放置24 h、自动进样器放置24 h、长期保存(-30℃放置28 d)的稳定性均良好。LC-MS/MS测定结果与室间质量评价靶值偏差为2.75%,斜率为1.04,相关系数(r2)为0.983。结论该LC-MS/MS采用放射性内标稀释,具有简单、快速、特异性和灵敏度较好的特点,连续6年测定结果符合全国室间质量评价要求,可用于茶碱的临床TDM。     

液相色谱-串联质谱检测血浆3-甲氧酪胺方法的建立

目的建立一种稳定的检测血浆3-甲氧酪胺(3-MT)的液相色谱-串联质谱(LC-MS/MS)方法。方法对LC-MS/MS的分离条件(色谱柱、柱温、p H值)进行优化,建立检测血浆3-MT的方法,并对该方法的线性范围、回收率、精密度、最低检测下限和稳定性进行评价。结果以同位素氘代作为内标,采用BEH HILIC色谱柱进行分离。流动相为100 mmol/L甲酸铵缓冲液(p H值为3)和纯乙腈,梯度洗脱;柱温为35℃。LC-MS/MS检测3-MT的线性范围为5~1 000 pg/mL,定量检出限为5 pg/mL,天间和批间变异系数(CV)分别为6%和7%,回收率为97.1%~109.3%。由于3-MT在室温中稳定性较差,因此需冰浴送检。结论建立了检测3-MT的LC-MS/MS方法,能够灵敏、准确地检测血浆3-MT水平。     

液相色谱-串联质谱法检测人血浆中碘帕醇

目的建立一种稳定的检测人血浆碘帕醇的液相色谱-串联质谱法(LC-MS/MS),并对该方法进行性能评价。方法以氘代同位素作为内标,人血浆样品经直接沉淀法前处理后进样,用XSelect^TM HSS PFP色谱柱进行分离,用含0.1%甲酸-5mmol/L九氟戊酸-10%乙腈的水溶液等度洗脱;正离子电喷雾离子化扫描检测,多反应监测(MRM)扫描分析,建立检测血浆碘帕醇的LC-MS/MS方法。根据CLSI EP10-A和CLSI C62-A,评价该方法的线性范围、正确度、精密度、选择性、基质效应、稳定性。结果 LC-MS/MS检测血浆碘帕醇的线性范围为1～1 000μgI/mL(以含碘量计算);低、中、高浓度(3、50、800μgI/mL)血浆质控品检测结果与理论靶值的偏移为-6.1%～7.5%,重复性以不精密度表示,CV<6.6%(4.6%～6.6%),期间精密度CV<8.5%(6.6%～8.5%),方法回收率为92.5%～95.9%,基质效应为88.8%～95.2%;方法选择性良好,溶血和脂血因素均不影响检测结果。血浆样品在室温下放置最好不要超过48 h,在-80℃下反复冻融不超过2次,经前处理后的血浆样品4℃自动进样器中放置不超过72 h,碘帕醇可稳定检测。结论建立并评价了一种可靠的检测人血浆碘帕醇的LC-MS/MS方法。     

LC-MS/MS检测血清万古霉素浓度方法的建立及评价

目的建立检测人血清中万古霉素浓度的液相色谱-串联质谱(LC-MS/MS)法,并与化学发光微粒子免疫法(CMIA)比较方法学性能。方法血清样品经过甲醇沉淀蛋白质,采用安捷伦Poroshell 120EC C18色谱柱(2.1 mm×50 mm,2.7μm)梯度洗脱分离,流动相为甲醇(0.1%甲酸)-水(0.1%甲酸),流速为0.5 m L/min,内标采用去甲万古霉素,质谱采用ESI正离子多反应监测扫描模式。用该方法定量检测112例临床服用万古霉素患者血清万古霉素含量,并与临床常用的CMIA所得结果进行比较。结果万古霉素在1~100μg/m L内线性关系良好(r2=0.996 4);方法日内日间精密度、准确度均满足药物定量检测要求,且无基质效应和残留效应。Wilcoxon符号秩和检验结果显示,LC-MS/MS测定结果与CMIA结果比较,差异有统计学意义(P0.05);相关性检验结果显示,两者线性相关性良好,Y=1.06X-0.37(r=0.986)。结论 LC-MS/MS法性能较好,价格优廉,与CMIA法有较好的相关性,且检测在准确度、精密度上优势明显,可用于临床检测万古霉素的血清药物浓度。     

同位素稀释液相色谱-串联质谱法检测血清睾酮候选参考方法的建立及应用

目的建立基于液相色谱串联质谱(LC-MS/MS)技术的血清睾酮候选参考方法,并采用该方法对临床常规检测方法(微粒子化学发光法)的正确性进行评价。方法采用睾酮-2,3,4-~(13)C_3作为内标,以等体积比的正己烷-乙酸乙酯溶液对样本进行液液萃取,上清液采用氮气吹干后用流动相复溶,进行LC-MS/MS分析。采用五点包括法计算血清睾酮浓度。参考美国临床实验室标准化协会(CLSI)C62-A文件和EP15-A3文件对建立的同位素稀释液相色谱-串联质谱(ID-LC-MS/MS)方法进行性能评价(方法残留、特异性、线性范围、精密度、准确度),参考CLSI EP9-A3文件对微粒子化学发光法的正确性进行评价。结果建立的ID-LC-MS/MS方法检测血清睾酮的分析时间为5min,特异性好,无残留,线性范围为0.034～22.00ng/mL,批内不精密度≤2.3%,批间不精密度≤2.2%,测定有证参考物质SRM971(Level male)的相对偏移为0.2%,测定2017 RELA-A、2017 RELA-B样本的相对偏移分别为-2.9%、-3.3%,测定2017 RELA-A样本的不确定度为0.11 ng/mL。微粒子化学发光法与ID-LC-MS/MS的相关性较好(r=0.963),但偏差较大[总平均偏差为-24.4%,低浓度样本(≤1 ng/mL)平均偏差为-62.4%,高浓度样本(1 ng/mL)平均偏差为6.3%]。结论成功建立了检测血清睾酮的ID-LC-MS/MS候选参考方法。该方法的精密度、准确度均较好,且操作简单,分析时间短。     

高效液相色谱串联质谱法测定血清非结合雌三醇的前处理研究

目的优化高效液相色谱串联质谱法(LC-MS/MS)测定血清游离雌三醇(uE3)的前处理方法。方法以活性炭吸附血清为研究基质,通过加入已知量的雌三醇(E3)标准品及E3-2-3-4-13 C3内标,在LC-20AD XR高效液相色谱系统和API 5500串联四级杆质谱仪上对E3检测的色谱条件、质谱条件及萃取条件等进行优化。结果色谱条件:选用菲罗门Knietex色谱柱(100.0mm×2.1mm,2.6μm);有机相为甲醇,水相为0.1mmol/L氟化铵水溶液,8min梯度洗脱。质谱条件:选用电喷雾(ESI)负离子模式和多反应监测(MRM)模式分析,选择质荷比(m/z)287→m/z 145作为E3的定量离子对,m/z 287→m/z 171作为定性监测离子对;选择m/z 290→m/z 148作为内标的定量离子对,m/z 290→m/z 174作为定性监测的离子对。萃取条件:萃取剂为正己烷/乙酸乙酯(50/50,v/v),样品与萃取剂的比例为1∶2,萃取率可达85.71%。结论 E3检测的色谱条件、质谱条件、萃取条件已得到全面优化,为后续建立LC-MS/MS测定人血中uE3的参考方法打下良好基础。     

一种免疫分析系统与液相色谱-串联质谱检测25-羟维生素D的一致性评价

目的:比较临床常用的罗氏全自动免疫分析系统（评估方法）与液相色谱-串联质谱（LC-MS/MS,参比方法）检测25-羟基维生素D［25（OH）D］的一致性。方法:收集2019年5—6月解放军总医院第二医学中心及第五医学中心健康查体剩余血清标本909份,分别由评估方法及LC-MS/MS检测25（OH）D。用Passing-...     

Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice

BACKGROUND: Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory. METHODS: We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h. RESULTS: Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. CONCLUSIONS: The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.     

Discordant carbamazepine values between two immunoassays: carbamazepine values determined by ADVIA Centaur correlate better with those determined by LC-MS/MS than PETINIA assay

Discordant carbamazepine values as determined by two different immunoassays may be due to different cross-reactivities with the active metabolite carbamazepine 10, 11-epoxide and may cause confusion in interpreting carbamazepine serum levels. In this study, we compared carbamazepine values in samples containing carbamazepine and the epoxide metabolite, as determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and by two commercial carbamazepine immunoassays: the PETINIA and the ADVIA Centaur carbamazepine. Clinical specimens were used for the comparative studies wherein we determined carbamazepine concentrations using the PETINIA, ADVIA Centaur, and LC-MS/MS assays. We observed an excellent correlation between carbamazepine concentrations determined by the ADVIA Centaur and LC-MS/MS methods while carbamazepine values were overestimated using the PETINIA assay. When aliquots of drug-free serum were supplemented with clinically relevant concentrations of the carbamazepine epoxide metabolite, we observed negligible cross-reactivity of epoxide with the ADVIA Centaur assay but over 90% cross-reactivity with the PETINIA assay. We conclude that the ADVIA centaur assay accurately measures carbamazepine concentrations in plasma or serum and that the PETINIA assay significantly overestimates true carbamazepine concentration. Such discordance may cause confusion in interpreting serum carbamazepine levels.     

Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma

BACKGROUND: Estradiol (E2) and estrone (E1) measurements form an integral part of the assessment of female reproductive function and have expanding roles in other fields. However, many E1 and E2 immunoassays have limited functional sensitivity, suffer from cross-reactivity, and display poor intermethod agreement. To overcome these problems, we developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous measurement of E1 and E2. METHODS: After dansyl chloride derivatization, samples were separated by fast gradient chromatography and injected into a tandem mass spectrometer after formation of positive ions with atmospheric pressure chemical ionization. The limits of detection and quantification, recovery, linearity, precision, and reference intervals were determined, and performance was compared with several immunoassays. RESULTS: Total run time per sample was 5 min. The multiple-reaction monitoring ion pairs were m/z 506/171 for 3-dansyl-estradiol and m/z 504/171 for 3-dansyl-estrone. The limits of detection for E1 and E2 were 12.9 pmol/L (3.5 ng/L) and 10.3 pmol/L (2.8 ng/L), respectively. Interassay imprecision (CV) was 4-20% (n = 20). The limits of quantification (functional sensitivities) for E1 and E2 were 44.1 pmol/L (11.9 ng/L) and 23.2 pmol/L (6.3 ng/L), respectively. The assay was linear to >2200 pmol/L ( approximately 600 ng/L) for each analyte. Recoveries were 93-108% for E1 and 100-110% for E2. No cross-reactivity was observed. Method comparison with several immunoassays revealed that the latter were inaccurate and prone to interferences at low E1 and E2 concentrations. CONCLUSIONS: LC-MS/MS allows rapid, simultaneous, sensitive, and accurate quantification of E1 and E2 in human serum.     

Comparison between a liquid chromatography-tandem mass spectrometry assay and a fluorescent polarization immunoassay to measure whole blood everolimus concentration in heart and renal transplantations

Various methods [fluorescent polarization immunoassay (FPIA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay] are used for therapeutic drug monitoring of everolimus. The aim of this study is to compare these assays in renal and heart transplantation. The correlation between results was investigated by linear regression in 44 patients (24 heart recipients and 20 renal recipients--137 samples). The comparison between assays was performed by a paired t-test. A highly significant correlation was found between FPIA and LC-MS/MS in heart and renal recipients [FPIA=0.851 x LC-MS/MS+1.773r(2)=0.8738 (P<0.001)]. Paired t-tests did not show a significant difference between everolimus whole blood concentrations in the populations of heart and renal recipients or heart recipients or renal recipients. FPIA and LC-MS/MS assays gave consistent overall results although some significant differences were observed in some samples between these methods indicating that FPIA assay has limitations that deserve further investigations.     

串联质谱和高效液相色谱-串联质谱二次筛查联合应用于新生儿MMA筛查

目的探讨串联质谱和高效液相色谱-串联质谱二次筛查联合应用在甲基丙二酸血症(MMA)中的筛查价值。方法收集新生儿串联质谱初筛结果中C3、C3/C2、C3/C0单一或多个指标异常的新生儿干血滤纸片标本,用高效液相色谱-串联质谱的方法定量检测原始血片中甲基丙二酸、甲基枸橼酸和高半胱氨酸,对二次筛查后疑似阳性的新生儿进行召回复查,并进行尿气相色谱/质谱检测。临床诊断患儿进一步予以基因检测进行确诊。结果共收集423例C3、C3/C2、C3/C0单一或多个指标异常的新生儿筛查标本,初筛阳性率约为1%,行联合筛查检测结果发现8例标本中甲基丙二酸和同型半胱氨酸表达水平明显升高,召回复查尿气相色谱质谱提示甲基丙二酸轻度升高。结论串联质谱和高效液相色谱-串联质谱联合应用可以提高新生儿MMA筛查的阳性预测值、降低假阳性率,在新生儿遗传代谢病筛查中具有重要价值。     

Quantitative, highly sensitive liquid chromatography-tandem mass spectrometry method for detection of synthetic corticosteroids

BACKGROUND: Measurements of serum or urine concentrations of synthetic glucocorticoids are useful for assessing suspected iatrogenic hypothalamic-pituitary-adrenal axis suppression and Cushing syndrome. We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantitative analysis of beclomethasone dipropionate, betamethasone, budesonide, dexamethasone, fludrocortisone, flunisolide, fluorometholone, fluticasone propionate, megestrol acetate, methylprednisolone, prednisolone, prednisone, triamcinolone, and triamcinolone acetonide. METHODS: Stable isotopes of cortisol-9,11,12,12-d(4) and triamcinolone-d(1) acetonide-d(6) were added as internal standards to calibrators, controls, and unknown samples. After acetonitrile precipitation, these samples were extracted with methylene chloride, and the extracts were washed and dried. Reconstituted extract (15 muL) was injected on a reversed-phase column and analyzed by LC-MS/MS in positive-ion mode. Assay precision, accuracy, linearity, and sample stability were determined by use of enriched samples. Clinical validation included analysis of 8 serum and 20 urine samples from patients with undetectable cortisol concentrations and analysis of different types of tablets. RESULTS: Functional assay sensitivity was as low as 0.6-1.6 nmol/L for all compounds except for triamcinolone (7.6 nmol/L). Interassay CVs were 3.0-20% for concentrations of 0.6-364 nmol/L for all analytes. Recoveries of all analytes (except triamcinolone in serum) were 82-138% at 19.2-693 nmol/L. All but one of the serum and urine samples from patients who were tested because of suppressed cortisol concentrations contained at least one synthetic steroid. Tablet analysis recovered 75% of the synthetic steroids in suspected drugs. CONCLUSIONS: LC-MS/MS allows simultaneous quantitative detection of various synthetic steroids in serum, plasma, urine, and tablets. This provides a valuable tool for evaluating the clinical effects of topical and systemic synthetic corticosteroids.     

Measurement of folate in fresh and archival serum samples as p-aminobenzoylglutamate equivalents

BACKGROUND: The development of accurate and precise folate assays has been difficult, mainly because of folate instability. Large interassay and interlaboratory differences have been reported. We therefore developed a serum folate assay that measures folate and putative degradation products as p-aminobenzoylglutamate (pABG) equivalents following oxidation and acid hydrolysis. METHODS: Serum was deproteinized with acid in the presence of 2 internal calibrators ([13C2]pABG and [13C5]5-methyltetrahydrofolate). 5-Methyltetrahydrofolate and other folate species in serum were converted to pABG by oxidation and mild acid hydrolysis. pABG and its internal calibrators were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The limit of quantification was 0.25 nmol/L, and the assay was linear in the range 0.25-96 nmol/L, which includes the 99.75 percentile for serum folate concentrations in healthy blood donors. Within- and between-day imprecision was < or = 5%. We detected no residual folate in serum samples after sample preparation. Folate concentrations in fresh serum samples obtained with the pABG assay and with a microbiologic assay showed good agreement (r = 0.96). In stored samples containing low folate concentrations due to folate degradation, the pABG assay yielded substantially higher folate concentrations than the microbiologic assay. CONCLUSIONS: The pABG assay combines automated sample preparation with LC-MS/MS analysis. It allows measurement of folate not only in fresh samples of serum/plasma but also in stored samples in which the folate has become oxidized and degraded to an extent that it cannot be assayed with traditional folate assays.     

Quantification by liquid chromatography tandem mass spectrometry of mycophenolic acid and its phenol and acyl glucuronide metabolites

BACKGROUND: We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure for the quantification of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites. METHODS: We performed protein precipitation on all samples (calibrators, quality controls, and patient samples) and then subjected them to online solid-phase extraction followed by reversed-phase liquid chromatography for 4.0 min. The carboxybutoxy ether of MPA (MPAC) was used as the internal calibrator. The separated compounds (MPA, MPAG, AcMPAG, and MPAC) were detected by electrospray ionization-coupled MS/MS. We compared LC-MS/MS results with results for the same samples obtained with a validated HPLC procedure with an ultraviolet detector. RESULTS: Comparison with the validated HPLC-ultraviolet procedure demonstrated good agreement. The Passing-Bablok regression was y = 0.968x - 0.058 for MPA, y = 1.08x - 1.697 for MPAG, and y = 0.952x + 0.076 for AcMPAG. Assay imprecision showed a CV <10% at 3 concentrations for each compound. The lower limit of quantification was 0.1 mg/L for MPA, 1.0 mg/L for MPAG, and 0.05 mg/L for AcMPAG. The mean analytical recovery was 90%-110%. The assay was linear from 0.1 to 50 mg/L for MPA (r = 0.9987), from 1 to 500 mg/L for MPAG (r = 0.9999), and from 0.05 to 10 mg/L for AcMPAG (r = 0.9988). Quantification of the compounds was not affected by in-source fragmentation or ion suppression. CONCLUSION: The LC-MS/MS assay described here is valid and reliable for the quantification of total MPA, MPAG, and AcMPAG in serum.     

Therapeutic monitoring of serum digoxin for patients with heart failure using a rapid LC-MS/MS method

ObjectiveHere we develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of digoxin in serum.Design and methodsThe serum samples were extracted with methyl tert-butyl ether using an isotope-labeled digoxin-d3 as internal standard. The analyte was separated on a reverse phase Capcell C18 column and detected in positive electrospray ionization multiple reaction monitoring mass spectrometry.ResultsThe chromatographic analysis was carried out within 3 min, but the complete analysis took longer because of the liquid–liquid extraction. The lower limit of quantification was 0.1 ng/mL for digoxin. The intra- and inter-batch precisions were less than 12%, and the bias ranged from ? 9.1% to 10.7%. The external quality assessment (EQA) results obtained with the LC-MS/MS method were comparable to target values. Subsequently, this method has been applied to the therapeutic monitoring of digoxin in a clinical setting.ConclusionIn this study, we have developed a rapid and reliable LC-MS/MS method for the therapeutic monitoring of digoxin in human serum.