Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid

BACKGROUND: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. METHODS: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. RESULTS: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA. CONCLUSIONS: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.     

Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry

BACKGROUND: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. METHODS: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. RESULTS: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. CONCLUSIONS: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.     

Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest. METHODS: We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6 min. RESULTS: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97-107%. We measured 8-72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327 nmol/L and 23 to 363 nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0-2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53-90% loss of the non-methylTHF fraction. CONCLUSION: This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.     

Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone

BACKGROUND: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. METHODS: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d(4) to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 microL of the reconstituted extract was injected onto a reversed-phase C(18) column and analyzed using a tandem mass spectrometer operating in the positive mode. RESULTS: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 microg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 microg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 microg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 microg cortisol/24 h (r(2) = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 microg cortisol/24 h (r(2) = 0.67; n = 99). CONCLUSION: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.     

Rapid micro-scale assay for homocysteine by liquid chromatography-tandem mass spectrometry

OBJECTIVES: Increased total homocysteine (tHcy) level is an independent risk factor for atherosclerosis and cardiovascular disease. Here, we describe a rapid tHcy micro-scale assay. METHODS: We developed an easy sample preparation and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of tHcy in human serum and plasma, respectively. RESULTS: The tHcy assay was linear up to 61.6 micromol/L, the detection limit was 1.0 micromol/L, the lower limit of quantification was 1.8 micromol/L, the imprecision in the range of 9.30-25.9 micromol/L was less than 5.9% and the analytical recovery was 94.7 +/- 6.3%. CONCLUSIONS: Our novel tHcy LC-MS/MS assay is a quick, precise and robust method for tHcy determination in routine diagnostics.     

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.     

Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry

BACKGROUND: Determination of nicotine metabolism/pharmacokinetics provides a useful tool for estimating uptake of nicotine and tobacco-related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine dependency treatment. METHODS: We developed a sensitive method for analysis of nicotine and five major nicotine metabolites, including cotinine, trans-3'-hydroxycotinine, nicotine-N'-oxide, cotinine-N-oxide, and nornicotine, in human urine by liquid chromatography coupled with a TSQ Quantum triple quadrupole tandem mass spectrometer (LC/MS/MS). Urine samples to which deuterium-labeled internal standards had been added were extracted with a simple solid-phase extraction procedure. Anabasine, a minor tobacco alkaloid, was also included. RESULTS: The quantification limits of the method were 0.1-0.2 microg/L, except for nicotine (1 microg/L). Cotinine-N-oxide, trans-3'-hydroxycotinine, nicotine, and anabasine in urine were almost completely recovered by the solid-phase extraction, whereas the mean extraction recoveries of nicotine-N'-oxide, cotinine, and nornicotine were 51.4%, 78.6%, and 78.8%, respectively. This procedure provided a linearity of three to four orders of magnitude for the target analytes: 0.2-400 microg/L for nicotine-N'-oxide, cotinine-N-oxide, and anabasine; 0.2-4000 microg/L for cotinine, nornicotine, and trans-3'-hydroxycotinine; and 1.0-4000 microg/L for nicotine. The overall interday method imprecision and recovery were 2.5-18% and 92-109%, respectively. CONCLUSIONS: This sensitive LC/MS/MS procedure can be used to determine nicotine metabolism profiles of smokers, people during nicotine replacement therapy, and passively exposed nonsmokers. This method avoids the need for a time-consuming and labor-intensive sample enrichment step and thus allows for high-throughput sample preparation and automation.     

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.     

High-throughput plasma docetaxel quantification by liquid chromatography-tandem mass spectrometry

BackgroundThe most valuable treatment option for breast, prostate and lung carcinomas is at present represented by a low dose of docetaxel, administered on a weekly basis. A better understanding of docetaxel pharmacokinetic and pharmacodynamic profiles could lead to an improvement in this dose regimen efficacy.MethodsIn this study a high-throughput method is described for the rapid quantification of docetaxel for large clinical pharmacology investigations. This analytical approach is based on an automatic on-line purification and enrichment technique followed by a measurement in tandem mass spectrometry through Multiple Reaction Monitoring.ResultsThe assay was validated over a 0.15–1500 ng/mL range. Intra-day precision ranged from 1.9% to 6.4%, while the inter-day was between 7.6% and 11.2%. The mean deviation from the nominal value ranged from ? 0.5% to 5.6% for the intra-day, and from ? 0.4% to 3.1% for the inter-day assay. Clinical applicability was demonstrated by measuring plasma pharmacokinetics in patients receiving weekly 25–35 mg/m² of docetaxel.ConclusionThe proposed LC–MS/MS assay was found to have a better performance than previously reported methods in terms of sensitivity and sample preparation. It does not require any laborious pre-analytical manipulation and can be easily employed in large clinical pharmacology studies.     

Sample preparation for liquid chromatography-tandem mass spectrometry using functionalized ferromagnetic micro-particles

ObjectivesTo study the applicability of functionalized ferromagnetic micro-particles in the sample preparation for quantification of small molecules by LC-MS/MS.Design and methodsA representative analyte (itraconazole) was extracted from plasma samples using small quantities of C18-modified ferromagnetic micro-particles.Results125 μg of functionalized micro-particles was sufficient to extract the target analyte from 10 μL of plasma with a recovery rate of approximately 100%; linearity (r > 0.99) and reproducibility (inter-assay CV ≤ 7%) of quantitative results was found.ConclusionsC18-functionalized ferromagnetic particles can be used to efficiently extract small molecule analytes from complex biological matrices for quantitative analyses using LC-MS/MS.     

液相色谱串联质谱技术在病原菌鉴定中的初步应用

摘要：目的 初步评估应用液相色谱串联质谱技术（ＬＣ ＭＳ ／ ＭＳ）对病原菌进行鉴定的可行性，为该技术临床应用提供实验依 据。方法 以全基因基础上的蛋白质组学库作为通用细菌鉴定数据库，用ＬＣ ＭＳ ／ ＭＳ技术对 ４１ 株标准菌株以及临床分离的 ２２ 株奈瑟菌和 ４６ 株大肠埃希菌进行鉴定。结果 串联质谱技术鉴定病原菌在种的水平鉴定准确率为 １００％。以 １３ 株标准 菌株样品稀释倍数和检测获得的肽段数量进行线性分析发现，二者之间具有很好的线性关系，相关系数均在 ０．９０ 以上。结论 ＬＣ ＭＳ ／ ＭＳ 有望成为病原菌鉴定检测新方法。     

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.     

Measurement of cystine in granulocytes using liquid chromatography-tandem mass spectrometry

BACKGROUND: Cystinosis is a rare autosomal recessive disorder characterized by an accumulation of intralysosomal cystine due to a defect in cystine transport across the lysosomal membrane. This disorder can be treated specifically using high doses of cysteamine. Accurate measurement of intracellular cystine content is necessary for the diagnosis and monitoring of treatment with cysteamine. Here we describe a new method to measure intracellular cystine. It relies on a liquid chromatography-tandem mass spectrometry assay. We compare this novel method with the cystine-binding protein assay. METHOD: Cells were isolated and lysed in the presence of N-ethylmaleimide to avoid interference from cysteine. After deproteinization, addition of stable isotope d6 cystine and butylation, cystine was measured using an API 3000 MSMS. RESULTS: The cystine assay was linear to at least 50 micromol/L. Within-run and between-run coefficients of variation were 2.9% and 5.7% respectively. CONCLUSION: It is possible to measure very low concentrations of intracellular cystine with liquid chromatography-tandem mass spectrometry. The results obtained with this novel method correlate very well with those obtained using the cystine-binding protein assay.     

Therapeutic drug monitoring of topiramate by liquid chromatography-tandem mass spectrometry

BackgroundTopiramate (TPM) is a new antiepileptic drug (AED) used worldwide in patients with various types of epilepsies and also for prophylaxis of migraine. A rapid, selective, reliable, precise, accurate, and reproducible tandem mass spectrometric (MS/MS) method for quantification of TPM in human plasma using topiramate-d₁₂ as an internal standard (IS) has been developed and validated to be used routinely for TDM of TPM.MethodsThe drug and IS were extracted by ether and analyzed on Symmetry^® C₁₈ column. Quantitation was achieved using ESI-interface employing MRM mode.ResultsThe method was validated over the concentration range of 0.5–30 µg/ml (r > 0.99). Intra- and inter-run precisions of TPM assay at three concentrations ranged from 0.7 to 7.8% with accuracy (bias) varied from ? 10.0 to 2.1% indicating good precision and accuracy. Analytical recoveries of TPM and IS from spiked human plasma were in the range of 84.1 to 90.0% and 90.0 to 111.0%, respectively. Stability of TPM in human plasma samples at different conditions showed that the drug was stable under the studied conditions. Matrix effect study showed a lack of matrix effect on mass ions of TPM and IS.ConclusionThe described method compared well when assessed by Heathcontrol TDM theme program (r > 0.99). The suitability of the developed method for TDM was demonstrated by measuring TPM in human plasma samples of epileptic patients treated with TPM. The proposed method is appropriate for routine TDM of TPM.     

Determination of urinary free cortisol by liquid chromatography-tandem mass spectrometry

Measurement of urinary free cortisol is clinically important in the diagnosis of Cushing's syndrome. While liquid chromatography (LC) with UV detection provides much better specificity than immunologic methods, certain drugs cause interference. Detection by mass spectrometry (MS) is a potentially superior method. Our analysis utilizes 1 mL urine spiked with 6-alpha-methylprednisolone as internal standard. The samples were extracted with dichlormethane and the extract was washed, evaporated to dryness and analyzed by LC-MS/MS operating in the negative mode after separation on a reversed-phase C18 column. The calibration curves for analysis of urinary cortisol exhibited consistent linearity and reproducibility in the range of 10-400 nmol/L. Inter-assay CVs were 4.0-7.6%, at mean concentrations of 21-153 nmol/L. The detection limit was 1 nmol/L (signal-to-noise ratio=3). The mean recovery of cortisol added to urine ranged from 67% to 87% and that of the internal standard from 71% to 76%. The regression equation for the LC-MS/MS (x) and HPLC (y) methods was: y=1.095x+8.0 (r=0.996; n=111). Drugs known to interfere with UV detection did not cause problems here. The sensitivity and specificity of the MS/MS method for urinary free cortisol offer advantages over HPLC with UV detection by eliminating drug interference. The higher equipment costs in comparison with HPLC methods using UV detection are balanced by higher throughput, thanks to 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.