Short-term biological variation and Reference change values of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine

AimThe DNA and RNA oxidative damage products urinary 8‐oxo‐7,8‐dihydro‐2''‐deoxyguanosine (8‐odGsn) and 8‐oxo‐7,8‐dihydroguanosine (8‐oGsn) have potential use in clinical practice. However, biological variation (BV) and reference change values (RCVs) have not been established. The aim of this study was to establish the short‐term between‐subject BV(CV_G)_, within‐subject BV(CV_I), and RCVs for urinary 8‐odGsn and 8‐oGsn.MethodsFirst‐morning midstream urine specimens were collected from 20 apparently healthy subjects(ten males and ten females) on five consecutive days. 8‐odGsn and 8‐oGsn were measured using LC‐MS/MS, while urine creatinine (U‐Cr) was also measured to correct their results. A two‐level nested ANOVA was used to estimate the CV_I and CV_G. ResultsThe values of CV_G for 8‐odGsn, 8‐odGsn/U‐Cr, 8‐oGsn, and 8‐oGsn/U‐Cr were 31.2%, 39.6%, 35.3%, and 28.8%, respectively, while CV_I for them were 40.5%, 9.0%, 33.5%, and 12.1%, respectively. The RCVs for 8‐odGsn, 8‐odGsn/U‐Cr, 8‐oGsn, and 8‐oGsn/U‐Cr were 112.5%, 26.7%, 93.7%, and 36.5%, respectively.ConclusionBV and RCVs were firstly established for 8‐oxo‐dGsn and 8‐oGsn, and can be used in clinical practice.     

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     

Determination of itraconazole and hydroxyitraconazole in plasma by use of liquid chromatography-tandem mass spectrometry with on-line solid-phase extraction.

In this paper a method for the simultaneous quantification of the anti-fungal drug itraconazole and its co-active metabolite hydroxyitraconazole in plasma employing liquid chromatography tandem-mass spectrometry and automated solid-phase extraction is described. The method proved rugged, enables short turn-around times and is highly specific. Since there is growing evidence for the importance of therapeutic drug monitoring of itraconazole in the prophylaxis and treatment of invasive fungal infections, the method described here is of interest for a large number of tertiary care hospital laboratories.     

Feasibility of standardization of serum C-peptide immunoassays with isotope-dilution liquid chromatography-tandem mass spectrometry

BACKGROUND: Serum C-peptide concentrations reflect pancreatic function in different clinical and diagnostic settings; however, the utility of C-peptide testing is limited by the lack of standardized commercial immunoassays. Standardization can best be done by split-sample comparison with a hierarchically higher reference measurement procedure with a set of native sera. For serum peptides, isotope-dilution liquid chromatography-mass spectrometry (ID-LC/MS) is recommended as a reference measurement procedure. METHODS: We evaluated the analytical performance characteristics of an ID-LC/tandem MS procedure for measurement of serum C-peptide after a 2-step solid-phase extraction. To investigate the feasibility of this procedure for use in standardization, we also performed a method comparison with 3 representative commercial assays. RESULTS: The ID-LC/tandem MS procedure showed maximum within-run, between-run, and total CVs on dedicated sera (C-peptide concentrations, 1.6 and 4.0 mug/L) of 2.1%, 2.5%, and 2.9%, respectively; an accuracy of 94.6%-104.1%; a minimum trueness of 98.1% (95% confidence interval, 96.2%-100.0%), and limits of quantification and detection of 0.15 and 0.03 mug/L, respectively. Deming linear regression analysis of the method-comparison data showed that the immunoassays correlated well with ID-MS and were specific, but lacked intercomparability and trueness. We propose that the deficiencies can be resolved by recalibration on the basis of the method comparison. CONCLUSIONS: The ID-LC/tandem MS procedure is suitable for specific and accurate measurement of basal and stimulated serum concentrations of proinsulin C-peptide fragment 33-63 and is suitable for use in standardization of C-peptide immunoassays.     

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.     

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 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.     

Quantification of urinary 8-iso-PGF2alpha using liquid chromatography-tandem mass spectrometry and association with elevated troponin levels

OBJECTIVES: Increased lipid peroxidation (i.e. "oxidative stress") has been identified as a central mechanism in the development of atherosclerosis and inflammatory vascular damage. Measurement of 8-iso-PGF(2alpha) has demonstrated to be a reliable indicator of in vivo oxidative stress levels. The purpose of this study was to develop a rapid, sensitive, and specific LC-MS/MS method for detection of urinary 8-iso-PGF(2alpha), establish reference intervals, and correlate isoprostane levels with cardiac troponin I. DESIGN AND METHODS: Urinary 8-iso-PGF(2alpha) was detected after direct injection onto a C18 silica column and monitored in the MRM mode using m/z transitions of 353.2>193.25 (8-iso-PGF(2alpha)) and 357.2>197.25 (8-iso-PGF(2alpha)-d(4)). The LC-MS/MS method was also compared to an ELISA kit. Reference interval studies were evaluated against a separate population of patients presenting with chest pain that had positive cTnI values. RESULTS: Elution of 8-iso-PGF(2alpha) was achieved after 7 min, with a total run time of 10 min. Inter-assay CVs were 13.8-20.0% and intra-assay CVs were 10.9-17.0%. Linearity ranged from 100 pg/mL to 100 ng/mL. Deming regression of ELISA and LC-MS/MS methods for 8-iso-PGF(2alpha) levels yielded poor correlation, with a slope of 0.0265, y-intercept of 0.255 ng/mL, and R(2) value of 0.0434. Urine 8-iso-PGF(2alpha) concentrations in samples obtained from healthy individuals (n=34) ranged from 57 to 390 ng/g creatinine with a mean of 221 ng/g creatinine. 8-iso-PGF(2alpha) levels were statistically significant in troponin-positive (n=35) versus troponin-negative (n=36) patients (p<0.0049). CONCLUSIONS: This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 8-iso-PGF(2alpha) in urine. 8-iso-PGF(2alpha) has potential to be a great prognostic risk indicator in individuals with a high probability for future coronary events.     

8-Oxo-7,8-dihydro-2'-deoxyguanosine Forms a Relatively Unstable Tetrameric Structure Compared with 2'-Deoxyguanosine

The hydrogen-bonded guanine tetrad, or G-quartet has been implicated in a variety of biological roles, including the function of chromosome telomeres. Here effect of the hydroxylation of guanosine at the 8 position on the G-quartet formation was examined. Electrospray inonization mass (ESI-MS) spectra of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 2'-deoxyguanosine (dG) were measured in order to know whether or not 8-oxodG forms a tetrameric structure as 2'-deoxyguanosine forms in teromeres. The ESI-MS spectra of dG shows prominent peaks at m/z 290, m/z 557, and m/z 1092, corresponding to [dG + Na]⁺, [dG2 + Na]⁺, and [dG₄ + Na]⁺ in the presence of 0.1 mM NaCl. On the other hand, the ESI-MS spectra of 8-oxodG in the presence of 0.1 mM NaCl shows prominent peaks at m/z 306 and m/z 589, corresponding to [8-oxodG + Na]⁺ and [8-oxodG₂ + Na]⁺. The results showed that 8-oxodG forms a relatively unstable tetrameric structure compared with dG.     

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.     

Quantification of urinary oxalate by liquid chromatography-tandem mass spectrometry with online weak anion exchange chromatography

BACKGROUND: Urinary oxalate is commonly measured with an enzymatic assay that is specific but requires a manual clean-up step to reduce ascorbic acid interference. We developed a urinary oxalate assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with anion exchange chromatography and simple sample preparation. METHODS: We added calibrator or urine sample (10 microL) to 10 microL of (13)C2 oxalate and 400 microL of water and performed separation on a Waters OASIS WAX column, flow rate 0.6 mL/min, and then elution for 0.3 min with water containing 2 mmol/L ammonium acetate and 1 mL/L formic acid and for 1.0 min with 750 mL/L methanol containing 20 mL/L ammonia. We detected multiple reaction monitoring transitions m/z 88.6 > 60.5 and m/z 90.5 > 61.5 for oxalic acid and 13C2-oxalate, respectively, with a Quattro micro tandem mass spectrometer in electrospray-negative mode. RESULTS: Oxalate and 13C2-oxalate eluted at 1.2 min. Mean recovery was 95%, limit of detection 3.0 micromol/L, lower limit of quantification 100.0 micromol/L, linearity to 2212 micromol/L, imprecision <6%, and bias <3% at 166, 880, and 1720 micromol/L. Oxalate eluted after the main area of ion suppression. Mean response ratios for urine and aqueous samples, enriched at 200 and 1000 micromol/L, were 3.7% and 5.4%, respectively. No interference was observed from other organic acids. Passing and Bablock regression analysis comparing the Trinity Biotech enzymatic reagent set and LC-MS/MS showed LC-MS/MS = 1.06 (enzymatic assay) -21.2, r = 0.964, n = 110. Bland Altman analysis showed general agreement, with a mean bias of -1.9 mumol/L. CONCLUSION: This LC-MS/MS assay is applicable for quantifying urinary oxalate excretion.     

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.     

A simple high-throughput method for the determination of plasma methylmalonic acid by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Cobalamin (Cbl) deficiency is a common clinical phenomenon, in particular among the elderly and possibly also among infants. Methylmalonic acid (MMA) is the most sensitive and specific marker of intracellular Cbl status, but its application is hindered by limited methods available for accurate and high-throughput MMA determination. METHODS: We developed a non-laborious method for determination of MMA without the need for prior derivatization using HPLC combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotope-labeled methyl-d(3)-malonic acid (MMA-d(3)) was added to 100 microL of plasma as an internal standard. After deproteinization by ultrafiltration, an acidified aliquot of the eluate was injected into the HPLC system and analyzed by LC-ESI-MS/MS monitoring of the carbonyl loss of MMA and MMA-d(3). RESULTS: Calibrations between 0.1 and 1.0 microM exhibited consistent linearity and reproducibility. The lower limit of detection for plasma MMA was 0.1 microM (signal-to-noise ratio > or = 10). The intra- and inter-assay CVs of ten determinations of a plasma sample were 1.5% and 6.7%, respectively, at a mean concentration of 0.29 microM. Inter-assay CVs for 25 determinations of low, medium and high concentrations (0.22, 0.45 and 0.94 microM MMA) were 8.3%, 5.9% and 4.6%, respectively. The mean recovery of MMA added to plasma was 100%. CONCLUSIONS: By avoiding derivatization, we developed a new, non-laborious, simple and reliable high-throughput method for the determination of MMA that is suitable for automation.