Quantification of sirolimus by liquid chromatography-tandem mass spectrometry using on-line solid-phase extraction.

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 microl) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6-50 microg/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 microg/l in routine use; the instrumentation proved to be reliable and required minimal maintenance. Clin Chem     

Clinical-scale high-throughput analysis of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine by isotope-dilution liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction

BACKGROUND: Quantification of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in urine or blood is used to assess and monitor oxidative stress in patients. We describe the use of on-line solid-phase extraction (SPE) and isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) for automated measurement of urinary 8-oxodGuo. METHODS: Automated purification of urine was accomplished with a switching valve and an Inertsil ODS-3 column. After the addition of 15N5-labeled 8-oxodGuo as an internal standard, urine samples were analyzed within 10 min without sample purification. This method was applied to measure urinary 8-oxodGuo in a group of healthy persons (32 regular smokers and 35 nonsmokers). Urinary cotinine was also assayed by an isotope-dilution LC-MS/MS method. RESULTS: The lower limit of detection was 5.7 ng/L on column (2.0 fmol). Inter- and intraday imprecision (CV) was < 5.0%. Mean recovery of 8-oxodGuo in urine was 99%-102%. Mean (SD) urinary concentrations of 8-oxodGuo in smokers [7.26 (3.14) microg/g creatinine] were significantly higher than those in nonsmokers [4.69 (1.70) microg/g creatinine; P < 0.005]. Urinary concentrations of 8-oxodGuo were significantly correlated with concentrations of cotinine in smokers (P < 0.05). CONCLUSIONS: This on-line SPE LC-MS/MS method is sufficiently sensitive, precise, and rapid to provide high-throughput direct analysis of urinary 8-oxodGuo without compromising quality and validation criteria. This method could be applicable for use in daily clinical practice for assessing oxidative stress in patients.     

Quantification of voriconazole in plasma by liquid chromatography-tandem mass spectrometry.

A convenient liquid chromatography-tandem mass spectrometry method for the quantification of the triazole antifungal agent voriconazole in plasma samples is described. Fenbuconazole is used as an internal standard. After protein precipitation, automated solid-phase extraction is applied. Electrospray ionization in the positive mode is used and the following mass transitions are recorded: voriconazole, 350-->127; and fenbuconazol, 337-->125. The analytical run time is 4 min. The response was linear from 78 to 5000 microg/L. The total coefficient of variation (n=16) was 12.6% for a low-concentration pool (143 microg/L), 4.7% for a medium-concentration pool (419 microg/L), and 5.0% for a high-concentration pool (4304 microg/L). The method is proposed for future investigations that should be performed to test the hypothesis that therapeutic drug monitoring of voriconazole is clinically useful.     

Determination of salivary cortisol by liquid chromatography-tandem mass spectrometry

Background: Late evening salivary cortisol concentrations are increasingly used as a screening test in suspected Cushing's syndrome partly because of easy sample collection. The cortisol immunoassays are prone to interference by cross-reacting steroids and therefore there is a need for improvement. The high specificity of an LC-MS assay provides a solution to the problem. Methods: Our liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis utilizes only 0.1 ml of saliva. The samples were extracted with dichloromethane. The extract was evaporated to dryness and cortisol was analysed by LC-MS/MS operating in the negative mode ESI after separation on a reversed-phase column. Results: The calibration curves for analysis of salivary cortisol exhibited consistent linearity and reproducibility in the range of 0.5–20nmol/L. Interassay CVs were 4.3–11% at cortisol concentrations of 0.6–14nmol/L. The lower limit of quantitation (LOQ) was 70pmol/L (signal to noise ratio=10). The mean recovery of the analyte added to saliva samples ranged from 95–106%. The upper limit of the reference range (95%) was 3.0nmol/L. Conclusions: Our method is rapid, sensitive and simple to perform with a routine LC-MS/MS spectrometer.     

Determination of serum amantadine by liquid chromatography-tandem mass spectrometry

BACKGROUND: Amantadine (1-adamantylamine) is used for treatment of influenza, hepatitis C, parkinsonism, and multiple sclerosis. Current amantadine analysis by HPLC or gas chromatography (GC) requires a laborious sample pretreatment with extraction and/or derivatization steps. We established an LC-MS/MS method without protein precipitation, centrifugation, extraction and derivatization steps. MATERIAL AND METHODS: 50 microl sample+50 microl of 0.4 mg/l 1-(1-adamantyl)pyridinium bromide as internal standard+1000 microl water (96-well plate). Of this 25 microl+500 microl water (96-well plate; final serum dilution 1:462). LC-MS/MS: Surveyor MS pump, Autosampler, triple-quadrupole TSQ Quantum mass spectrometer (Thermo Electron). Autosampling: 2 microl of each sample. Chromatography: isocratic water/acetonitrile (60/40 v/v) with 5 g/l formic acid, flow rate 0.2 ml/min, run time 3 min, Phenomenex Luna C8(2) (100 x 2.0 mm (i.d.); 3-microm bead size) column. Mass spectrometry: electrospray atmospheric pressure ionization, positive ion and selective reaction monitoring mode, ion transitions m/z 152.0-->135.1 (at 22 eV amantadine) and 214.1-->135.1 (at 26 eV internal standard). RESULTS: Calibration curves were constructed with spiked serum samples (amantadine 50-1000 microg/l, r>0.99). No carry over (5000 microg/l). No ion suppression with retention times similar to those of amantadine (1.8 min) and the internal standard (2.1 min). Detection limit 20 mg/l, linearity 20-5000 mg/l, intra-assay/inter-assay CV<6%/<8%, recovery 99-101%. Method comparison: LC-MS/MS=1.23 x GC-45 (Passing-Bablok regression). No significant bias between GC and LC-MS/MS (Bland-Altman plot). CONCLUSION: We consider the sample pretreatment without deproteination, derivatization and centrifugation steps and the specificity of the tandem mass spectrometry as the most important points of our amantadine analysis method.     

Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry

Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable‐isotope dilution liquid chromatography‐tandem mass spectrometric (LC‐MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether‐ethyl acetate from 250?µL or 500?µL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple‐reaction monitoring (MRM) mode after separation on a reversed‐phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d₂ testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2–100?nmol/L. Interassay CVs were 4.2–7.6?% at mean concentrations of testosterone of 3.3–45?nmol/L. Total measurement uncertainty (U, k = 2) was 12.9?% and 13.4?% at testosterone levels of 2.0?nmol/L and 20?nmol/L, respectively. The limit of detection was 0.05?nmol/L (signal‐to‐noise ratio = 3) and the overall method recovery of testosterone was 95?%. Correlation (r) with our in‐house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18–30, 31–50, 51–70 and over 70 years were established. Sensitivity and specificity of the LC‐MS/MS method offer advantages over immunoassay and make it suitable for use as a high‐throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.     

Determination of testosterone in serum by liquid chromatography-tandem mass spectrometry

Commercial direct immunoassays for serum testosterone sometimes result in inaccuracies in samples from women and children, leading to misdiagnosis and inappropriate treatment. The diagnosis of male hypogonadism also requires an accurate testosterone assay method. We therefore developed a sensitive and specific stable-isotope dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for serum testosterone at the concentrations encountered in women and children. Testosterone was extracted with ether-ethyl acetate from 250 microL or 500 microL of serum. Instrumental analysis was performed on an API 2000 tandem mass spectrometer in the multiple-reaction monitoring (MRM) mode after separation on a reversed-phase column. The MRM transitions (m/z) were 289/97 for testosterone and 291/99 for d(2) testosterone. The calibration curves exhibited consistent linearity and repeatability in the range 0.2-100 nmol/L. Interassay CVs were 4.2-7.6 % at mean concentrations of testosterone of 3.3-45 nmol/L. Total measurement uncertainty (U, k = 2) was 12.9 % and 13.4 % at testosterone levels of 2.0 nmol/L and 20 nmol/L, respectively. The limit of detection was 0.05 nmol/L (signal-to-noise ratio = 3) and the overall method recovery of testosterone was 95 %. Correlation (r) with our in-house extraction RIA was 0.98 and with a commercial RIA 0.92. Reference intervals for adult males and females in age groups 18-30, 31-50, 51-70 and over 70 years were established. Sensitivity and specificity of the LC-MS/MS method offer advantages over immunoassay and make it suitable for use as a high-throughput assay in routine clinical laboratories. The high equipment costs are balanced by higher throughput together with shorter chromatographic run times.     

Determination of serum cortisol by isotope-dilution liquid-chromatography electrospray ionization tandem mass spectrometry with on-line extraction.

A liquid chromatographic-mass spectrometric method for the determination of cortisol in serum using atmospheric pressure electrospray ionization and tandem mass spectrometry is described. During sample preparation, 150 microl of serum were deproteinized with methanol/zinc sulfate followed by on-line solid phase extraction employing column switching. Tri-deuterated cortisol was used as the internal standard. The following transitions were monitored: cortisol, 363>309 m/z; d3-cortisol, 366>312 m/z. The total run-time was 5 minutes. The method proved linear (0-500 microg/l; r=0.999), precise (total coefficient of variation between 5.0% and 3.2% at a mean cortisol concentration of 15.1 microg/l and 269 microg/l, respectively; n=16) and specific with regard to relevant endogenous and exogenous steroids.     

Determination of free tetrahydrocortisol and tetrahydrocortisone ratio in urine by liquid chromatography-tandem mass spectrometry

OBJECTIVE: Measurement of urinary free tetrahydrocortisol and tetrahydrocortisone ratio (allo-THF+THF)/THE is clinically important in the diagnosis of hypertension caused by congenital absence of 11beta-hydroxysteroid dehydrogenase type 2 (apparent mineralocorticoid excess, AME) or inhibition of the enzyme after licorice ingestion. Although gas chromatography-mass spectrometry (GC-MS) provides reliable results, it requires derivatization and is lengthy and time-consuming. The purpose of this study was to demonstrate that detection by liquid chromatography-mass spectrometry (LC-MS) is a potentially superior method. MATERIAL AND METHODS: The analysis utilizes 1 mL urine. The samples were extracted with solid-phase extraction (SPE) using ethyl acetate as eluent. The extract was evaporated to dryness, and allo-tetrahydrocortisol (allo-THF), THF and THE concentrations were analyzed by LC-MS/MS operating in the negative mode after separation on a reversed-phase column. The calibration curves exhibited consistent linearity and reproducibility in the range of 7.5-120 nmol/L. Interassay CVs were 7.0-10 % at mean ratios of (allo-THF+THF)/THE of 0.54-1.9. The detection limit of the analytes was 0.4-0.8 nmol/L (signal-to-noise ratio = 3). The mean recovery of the three analytes ranged from 88 to 95 %. The regression equation for the free ratio using the LC-MS/MS (x) method and the total ratio using the GC-MS (y) method was: y = 0.30x+0.91 (r = 0.61; n = 25). CONCLUSIONS: The sensitivity and specificity of the LC-MS/MS method offer an advantage over GC-MS by eliminating derivatization. The high costs of equipment are balanced by higher through-put, owing also to shorter chromatographic run times.     

羊水样本卵磷脂和鞘磷脂检测超高效液相色谱串联质谱方法的建立及应用

目的建立检测羊水中卵磷脂和鞘磷脂(L/S)的超高效液相色谱串联质谱(UPLC-MS/MS)方法,以便准确、高效地预测胎儿肺成熟度。方法收集孕32~39周孕妇分娩时的羊水样本23份。依据新生儿Apgar评分标准,有3例胎儿胎肺未成熟、20例胎儿胎肺成熟。另收集孕18周孕妇羊水样本7份作为基线对照,建立检测羊水中卵磷脂和鞘磷脂水平的UPLC-MS/MS方法,计算卵磷脂/鞘磷脂(L/S)比值,同时采用板层小体计数(LBC)法检测板层小体(LB),评价2种方法在预测胎肺成熟度中的价值。结果建立的检测羊水中卵磷脂和鞘磷脂的UPLC-MS/MS方法精密度良好,离子峰强度和保留时间均在可检测范围内,主成分分析(PCA)显示6个质控样本聚类良好。以L/S比值=10作为判断胎肺成熟度与不成熟的临界值,UPLC-MS/MS的敏感性和特异性均为100%。以LB=50×10^9/L作为判断胎肺成熟度与不成熟的临界值,LBC法的敏感性和特异性分别为80%和95%。结论建立了检测羊水中卵磷脂和鞘磷脂水平的UPLC-MS/MS方法,其结果可靠,可以准确、高效地预测胎肺成熟度。     

人血浆百草枯高效液相色谱-串联质谱法的建立与评价

目的建立测定人血浆百草枯浓度的高效液相色谱-串联质谱法并评价。方法用甲醇蛋白质沉淀法对血浆样品预处理后,以乙腈-水(含200 mmol/L甲酸铵和0.1%甲酸)为流动相梯度进样,流速为0.4 mL/min。通过色谱柱(Waters XBridge BEH HILIC,2.5μm,2.1 mm×100 mm)分离样品。以ESI正离子模式、多反应离子监测(MRM)模式监测百草枯(m/z 186.1→171.1作为定量离子对)。用该方法检测临床患者血浆百草枯浓度,用ROC曲线评价血浆百草枯浓度和百草枯中毒严重指数(SIPP)对临床结局的判断价值。结果血浆百草枯浓度在50～10 000 ng/mL时,线性关系良好(R~2=0.997),最低定量下限为50 ng/mL。低(100 ng/mL)、中(2 000 ng/mL)、高(8 000 ng/mL)3个浓度水平质控品的不精密度均符合要求,且无基质效应。处理后样品室温放置6 h内稳定,样本反复冻融3次对结果无影响。31例中毒患者SIPP为17.76(0.30～90.91)h·mg/L,死亡组SIPP高于存活组(P0.05)。SIPP的ROC曲线下面积为0.889,最佳临床临界值为11.679 h·mg/L。结论本法灵敏、准确、快速、特异性好,适用于临床检测百草枯中毒患者。     

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。