Sihe Wang

Development and validation of a liquid chromatography-tandem mass spectrometry assay for serum 25-hydroxyvitamin D2/D3 using a turbulent flow online extraction technology

Abstract Background: Vitamin D is important to health and disease. Liquid chromatography-tandem mass spectrometry (LC-MSMS) is considered the most accurate technology for quantification of serum 25-hydroxyvitamin D (25OHD) which is the best biomarker for estimating vitamin D nutritional status. Methods: Serum was mixed with acetonitrile containing hexadeuterated 25-hydroxyvitamin D3 (d6-25OHD3) and centrifuged 10 min at 15,634×g. The supernatant was injected onto a turbulent flow preparatory column then transferred to a polar endcapped C18 analytical column. The mass spectrometer was set for positive atmospheric pressure chemical ionization. Results: The analytical cycle time was 5.5 min. Inter- and intra-assay CV for both analytes across three concentrations ranged from 3.8% to 14.2%. The method was linear from 3.0 to 283.6 nmol/L for 25-hydroxyvitamin D3 (25OHD3) and 4.6 to 277.9 nmol/L for 25-hydroxyvitamin D2 (25OHD2), with an accuracy of 88.7%–118.7% and 90.7%–100.3%, respectively. No carryover or ion suppression was observed. Comparison with a radioimmunoassay using patient specimens (n=527) showed a mean difference of 5.2%, and diagnostic agreement of 80.9% with Deming regression of slope 0.867, intercept 12.8, standard error of estimate (SEE) 17.4, and r=0.8425. Conclusions: The LC-MSMS method coupled with turbulent flow technology for serum 25OHD quantitation is rapid, efficient, and suitable for clinical testing. Clin Chem Lab Med 2009;47:1565–72.

Positive newborn screen for methylmalonic aciduria identifies the first mutation in TCblR/CD320, the gene for cellular uptake of transcobalamin‐bound vitamin B12

Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin‐bound cobalamin (holo‐TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo‐TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low‐density lipoprotein receptor type A‐like domain. Inserting the codon by site‐directed mutagenesis fully restored TCblR function. Hum Mutat 31:1–6, 2010.

A novel HPLC method for quantification of 10 antiepileptic drugs or metabolites in serum/plasma using a monolithic column.

Therapeutic drug monitoring of antiepileptic drugs (AEDs) is important in maximizing the therapeutic response while minimizing the adverse effects. High-performance liquid chromatography (HPLC) is the most commonly used technique for this purpose. Recently, commercial monolithic columns were introduced, which consist of a single rod of fused silica or polymer. The objective of this work was to develop a simple and fast method to quantify 10 commonly measured AEDs or metabolites [carbamazepine, carbamazepine-10,11-epoxide, felbamate, lamotrigine, 10,11-dihydro-10-hydroxy-carbamazepine (active metabolite of oxcarbazepine), pentobarbital, phenobarbital, phenytoin, primidone, and zonisamide] in serum/plasma by HPLC using a reverse-phase monolithic column. Serum/heparin plasma (100 μL) was mixed with an internal standard solution (5-ethyl-5-p-tolylbarbituric acid in methanol, 250 μL). After centrifugation at 15,500g for 10 minutes, 15 μL of supernatant was injected into a monolithic column. The analytes were eluted with an isocratic solution of 0.1 M, pH 6.5, phosphate buffer:methanol:acetonitrile (77:20:3), monitored at 210 nm. The chromatography time was 16 minutes. The method was linear from 0.4-4.9 to 21.2-190.9 μg/mL depending on the analytes with analytical recovery of 80%-114%. The inter- and intra-assay coefficients of variation were <8% in 3 levels of serum-based controls for all the analytes. No significant carryover was observed. Commercial controls containing >100 therapeutic drugs and common endogenous substances were tested and showed no interference. Comparison studies for 6 AEDs or metabolites were performed against commercial HPLC methods. Three AEDs were compared with Food and Drug Administration-approved immunoassays. All comparisons had R > 0.96 with slope ranging from 0.86 to 1.20. This is a simple and fast HPLC method suitable for measuring the 10 AEDs or metabolites. The use of the monolithic column resulted in increased sensitivity, better resolution, and a shorter analytical time compared with a regular C18 column.

A rapid liquid chromatography-tandem mass spectrometry analysis of whole blood sirolimus using turbulent flow technology for online extraction

Abstract Background: Sirolimus is an immunosuppressant used in solid organ transplantation. Due to variable individual pharmacokinetics and narrow therapeutic ranges, therapeutic drug monitoring (TDM) is critical to the success of post-transplantation patient care. We developed a rapid method quantifying whole blood sirolimus using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an automated online extraction technology. Methods: Whole blood (100 μL) was mixed with a precipitation solution containing internal standard (32-desmethoxyrapamycin) and centrifuged at 15,634×g for 10 min. The supernatant (50 μL) was injected onto a turbulent flow preparatory column and then a C18 analytical column. The mass spectrometer was set for positive electrospray to monitor the ammonium adducts. Results: Analytical time was 4 min/injection. Inter- and intra-assay variation coefficients across three concentration levels ranged from 2.3% to 7.4%. The method was linear from 1.0 to 100.0 ng/mL with an accuracy of 93.3%–100.0%. No carryover was detected from samples at 313.6 ng/mL. There was no obvious ion suppression from patient samples or interference from other commonly used immunosuppressants. Good correlation with an in-house commercial LC-MS was observed. Conclusions: The LC-MS/MS method coupled with turbulent flow technology is rapid and efficient in TDM of whole blood sirolimus. Clin Chem Lab Med 2008;46:1631–4.

Simultaneous quantification of 19 drugs/metabolites in urine important for pain management by liquid chromatography-tandem mass spectrometry

Abstract Background: Monitoring pain management drugs and frequently abused drugs is important for physicians to assess patient compliance. Liquid chromatography-tandem mass spectrometry offers high specificity needed for this purpose. In this report, a novel liquid chromatography-tandem mass spectrometry method for simultaneously monitoring 19 drugs/metabolites in urine was developed and validated. Methods: Sample preparation included hydrolysis, dilution and turbulent flow online extraction. Analysis was achieved by reverse phase liquid chromatography and triple-quadruple tandem mass spectrometry. Two fragment ions, one for quantification and the other for assuring identification, were monitored for each analyte. Results: No matrix effect or interference was observed. Lower limits of quantification ranged from 5 to 25 ng/mL. Within the linear range, analytical recovery was between 85.8% and 119.4%. Intra-assay and total coefficient of variations were between 0.2% and 12.7%. This method was compared with mass spectrometry methods offered by two other laboratories using 82 patient samples and 60 spiked urine samples showing 60%–100% agreement. The current method identified more positive samples for all analytes except THCA. The discrepancy in detection rates was primarily due to the different cut-offs used by the other laboratories. Conclusions: A sensitive and specific liquid chromatography-tandem mass spectrometry method was developed for measuring 19 drugs/metabolites in urine important for pain management clinics.

THE MMACHC PROTEOME: HALLMARKS OF FUNCTIONAL COBALAMIN DEFICIENCY IN HUMANS

Cobalamin (Cbl, B(12)) is an essential micronutrient required to fulfill the enzymatic reactions of cytosolic methylcobalamin-dependent methionine synthase and mitochondrial adenosylcobalamin-dependent methylmalonyl-CoA mutase. Mutations in the MMACHC gene (cblC complementation group) disrupt processing of the upper-axial ligand of newly internalized cobalamins, leading to functional deficiency of the vitamin. Patients with cblC disease present with both hyperhomocysteinemia and methylmalonic acidemia, cognitive dysfunction, and megaloblastic anemia. In the present study we show that cultured skin fibroblasts from cblC patients export increased levels of both homocysteine and methylmalonic acid compared to control skin fibroblasts, and that they also have decreased levels of total intracellular folates. This is consistent with the clinical phenotype of functional cobalamin deficiency in vivo. The protein changes that accompany human functional Cbl deficiency are unknown. The proteome of control and cblC fibroblasts was quantitatively examined by two dimensional difference in-gel electrophoresis (2D-DIGE) and liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI/MS). Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, the neurological system and cell signaling. Pathway analysis of the differentially expressed proteins demonstrated strong associations with neurological disorders, muscular and skeletal disorders, and cardiovascular diseases in the cblC mutant cell lines. Supplementation of the cell cultures with hydroxocobalamin did not restore the cblC proteome to the patterns of expression observed in control cells. These results concur with the observed phenotype of patients with the cblC disorder and their sometimes poor response to treatment with hydroxocobalamin. Our findings could be valuable for designing alternative therapies to alleviate the clinical manifestation of the cblC disorder, as some of the protein changes detected in our study are common hallmarks of known pathologies such as Alzheimers and Parkinsons diseases as well as muscular dystrophies.

A simple liquid chromatography-tandem mass spectrometry method for measuring metanephrine and normetanephrine in urine

Abstract Background: Measuring urinary fractionated metanephrines is one of the initial tests in the diagnosis of pheochromocytoma. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) represents the most specific and accurate technology for this purpose. The goal of this work was to develop a simple LC-MS/MS method for measuring metanephrines in urine. Methods: Each urine sample was complexed with diphenylboronic acid, and purified on a Bond-Elute Plexa cartridge. The extract was concentrated and analyzed on a short Atlantis T3 column in 8.5 min. Metanephrines and their deuterated internal standards were monitored in positive electrospray ionization mode by multiple reaction monitoring. Results: Ion suppression was observed, but was compensated for by the respective internal standard. The analytical measurement range was 0.2–27.4 μmol/L and 0.3–14.6 μmol/L for metanephrine and normetanephrine, respectively. The intra-assay and total coefficient of variation throughout the linear ranges was 2.03%–2.11% and 2.20%–3.80% for metanephrine, and 4.50%–8.09% and 9.00%–10.00% for normetanephrine, respectively. Comparison with a commercial HPLC method using patient samples (n=65) by Passing-Bablok regression showed a slope of 1.000 and 1.014, y-intercept of –0.080 and –0.067, a correlation coefficient of 0.8830 and 0.9022, and a mean difference of 14.0% and –0.43% for metanephrine and normetanephrine, respectively. Conclusions: This simple method for urine metanephrines is suitable for clinical use.

Comparison of a stable isotope-labeled and an analog internal standard for the quantification of everolimus by a liquid chromatography-tandem mass spectrometry method.

Background: Everolimus is an immunosuppressant drug used in solid organ transplantation. Immunoassays and liquid chromatography–mass spectrometry (LC-MS) methods have been used for therapeutic drug monitoring of this drug. In LC–tandem mass spectrometry (MS/MS) methods, both 32-desmethoxyrapamycin and everolimus-d4 have been used as internal standards. Objectives: To compare 2 internal standards (32-desmethoxyrapamycin and everolimus-d4) for the quantification of everolimus by an LC-MS/MS method. Methods: Both 32-desmethoxyrapamycin and everolimus-d4 were introduced in the method validation process with 2 transitions simultaneously monitored for everolimus (975.6 → 908.7 as the quantifier and 975.6 → 926.9 as the qualifier) by an established LC-MS/MS method. The key performance characteristics were lower limit of quantification, accuracy, precision, and comparison with an LC-MS/MS method offered by another laboratory. Results: The lower limit of quantification (LLOQ) was 1.0 ng/mL using either internal standard with an analytical recovery of 98.3%–108.1% across the linear range. The total coefficient of variation for everolimus was 4.3%–7.2% with no significant difference between the 2 internal standards. In comparison with an independent LC-MS/MS method, though everolimus-d4 offered a better slope (0.95 versus 0.83), both internal standards showed acceptable results and had a coefficient of correlation r > 0.98 in the tested concentration range of 1.2–12.7 ng/mL. Conclusions: Although everolimus-d4 offered a more favorable comparison with an independent LC-MS/MS method, both everolimus-d4 and 32-desmethoxyrapamycin had acceptable performance as the internal standards for everolimus quantification by the LC-MS/MS method.

A Sensitive and Interference-Free Liquid Chromatography Tandem Mass Spectrometry Method for Measuring Metanephrines in Plasma

Background: Plasma metanephrines are the primary biomarkers used to aid in diagnosing pheochromocytoma. However, the low physiological levels of metanephrines, physicochemical properties, and potential interferences make it challenging to achieve high sensitivity and specificity. In this report, we developed and validated a sensitive and interference-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring plasma metanephrines with simple sample preparation. Methods: Plasma samples were extracted using weak cation exchange solid-phase extraction cartridges, and analyzed by LC-MS/MS with an analytical cycle time less than six minutes. Results: Absolute ion suppression and matrix effect were observed, however, were completely compensated for by the internal standards. Epinephrine, an isobaric interferent of normetanephrine, was chromatographically separated, and no interference was observed from other common interferents. The method was linear from 0.08 to 22.2 nmol/L for normetanephrine and 0.03 to 8.2 nmol/L for metanephrine with accuracy ranging from 81 to 107%. No carryover was observed up to 56.8 nmol/L for normetanephrine and 8.7 nmol/L for metanephrine. Intra-assay and total CVs were within 6.8% for normetanephrine and 5.2% for metanephrine for three levels tested. Based on Deming regression, comparison with a reference LC-MS/MS method using patient specimens (n=40) showed a slope of 0.973, intercept of 0.11 nmol/L and correlation coefficient of 0.9936 for normetanephrine and a slope of 1.039, intercept of -0.014 nmol/L, and correlation coefficient of 0.9914 for metanephrine. The mean difference was 3.5% and -1.6% for normetanephrine and metanephrine, respectively. Conclusion: This LC-MS/MS assay is sensitive and free of interference for quantitation of plasma metanephrines

A simple and robust LC–MS/MS method for measuring sirolimus and everolimus in whole blood

Therapeutic drug monitoring of immunosuppressants sirolimus and everolimus is mandatory and liquid chromatography tandem mass spectrometry (LC-MS/MS) is the preferred technology for the measurement. Due to the high hydrophobicity these analytes bind to reverse-phase columns tightly and need column heating to elute. Column heating not only requires extra instrument preparation but also causes permanent column damage if the heater is left on while elution pumps stop by the end of the run. The primary improvement in the current method was to elute the analytes at room temperature using special buffers. This new LC-MS/MS method has been validated for clinical use and offers improved simplicity and robustness by eliminating column heating yet with high sensitivity, precision and accuracy.