Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men,women, and children

BACKGROUND: Commercially available testosterone immunoassays give divergent results, especially at the low concentrations seen in women. We compared immunoassays and a nonimmunochemical method that could quantify low testosterone concentrations. METHODS: We measured serum testosterone in 50 men, 55 women, and 11 children with use of eight nonisotopic immunoassays, two isotopic immunoassays, and isotope-dilution gas chromatography-mass spectrometry (ID/GC-MS). RESULTS: Compared with ID/GC-MS, 7 of the 10 immunoassays tested overestimated testosterone concentrations in samples from women; mean immunoassay results were 46% above those obtained by ID/GC-MS. The immunoassays underestimated testosterone concentrations in samples from men, giving mean results 12% below those obtained by ID/GC-MS. In women, at concentrations of 0.6-7.2 nmol/L, 3 of the 10 immunoassays gave positive mean differences >2.0 nmol/L (range, -0.7 to 3.3 nmol/L) compared with ID/GC-MS; in men at concentrations of 8.2-58 nmol/L, 3 of the 10 immunoassays tested gave mean differences >4.0 nmol/L (range, -4.8 to 2.6 nmol/L). CONCLUSION: None of the immunoassays tested was sufficiently reliable for the investigation of sera from children and women, in whom very low (0.17 nmol/L) and low (<1.7 nmol/L) testosterone concentrations are expected.     

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.     

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.     

Determination of 17alpha-hydroxyprogesterone in serum by liquid chromatography-tandem mass spectrometry and immunoassay

17Alpha-hydroxyprogesterone (17OHP) is the most important serum marker for congenital adrenal hyperplasia (CAH). 17OHP is usually measured by immunoassay but its detection by mass spectrometry (MS) is a potentially superior method. An LC-MS (liquid chromatography-mass spectrometry) method was developed which utilizes 0.5 ml serum spiked with 6-alpha-methylprednisolone (6-MP) or deuterated 17OHP (d8-IS) as the internal standard. The samples were extracted with ether/ethylacetate, and the extract was evaporated to dryness and analysed by LC-MS/MS operating in the positive mode after separation on a reversed-phase C18 column. The calibration curves for analysis of serum 17OHP exhibited consistent linearity and reproducibility in the range of 5-250 nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23 nmol/l, respectively. The detection limit was 1 nmol/l (signal-to-noise ratio=3). The mean recovery of 17OHP added to serum ranged from 76 to 89% and that of internal standards from 75 to 82%. The regression equation for the LC-MS/MS (x) and in-house radioimmunoassay (RIA) (y) methods was: y=0.87x+0.26 (r=0.97; n=100) and for a commercial RIA it was: y=1.32x+0.02 (r=0.97; n=26).     

Low serum testosterone assayed by liquid chromatography-tandem mass spectrometry. Comparison with five immunoassay techniques

BACKGROUND: Low levels of serum testosterone, as typically found in women and children, cannot be measured reliably by immunoassays. Our aim was to develop a sensitive assay to quantitate low serum testosterone concentrations using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were compared to those obtained with various immunoassay techniques. METHODS: Serum testosterone levels in 70 women and children were measured using LC-MS/MS and compared with two automated, non-isotopic immunoassays, and three manual, isotopic immunoassays. Serum extraction was required only for LC-MS/MS and one of the isotopic methods. RESULTS: Deming regression analysis was used for comparison: the correlation coefficients were between 0.772 and 0.870, and the slopes between 0.972 and 1.365. Using Bland and Altman analysis, all the 5 immunoassays showed a positive mean difference compared with LC-MS/MS: all overestimated the testosterone levels in women and children. CONCLUSION: None of the immunoassays tested proved sufficiently reliable when low testosterone concentrations (< or =3.47 nmol/L) were measured. In contrast to conventional isotopic and non-isotopic immunoassay techniques, LC-MS/MS allows the precise determination of low testosterone levels. It has adequate sensitivity and is not subject to interference from other steroids that were tested.     

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.     

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.     

Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma

BACKGROUND: Metanephrines are biochemical markers for tumors of the adrenal medulla (e.g., pheochromocytoma) and other tumors derived from neural crest cells (e.g., paragangliomas and neuroblastomas). We describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of urinary conjugated metanephrines. METHODS: We added 250 ng of d3-metanephrine (d3-MN) and 500 ng of d3-normetanephrine (d3-NMN) to 1 mL of urine samples as stable isotope internal standards. The samples were then acidified, hydrolyzed for 20 min in a 100 degree C water bath, neutralized, and prepared by solid-phase extraction. The methanol eluates were analyzed by LC-MS/MS in the selected-reaction-monitoring mode after separation on a reversed-phase amide C16 column. RESULTS: Multiple calibration curves for the analysis of urine MN and NMN exhibited consistent linearity and reproducibility in the range of 10-5000 microg/L. Interassay CVs were 5.7-8.6% at mean concentrations of 90-4854 microg/L for MN and NMN. The detection limit was 10 microg/L. Recovery of MN and NMN (144-2300 microg/L) added to urine was 91-114%. The regression equation for the LC-MS/MS (x) and colorimetric (y) methods was: y = 0.81x - 0.006 (r = 0.822; n = 110). The equation for the HPLC (x) and LC-MS/MS (y) methods was: y = 1.09x + 0.05 (r = 0.998; n = 40). CONCLUSIONS: The sensitivity and specificity of the MS/MS method for urinary conjugated metanephrines offer advantages over colorimetric, immunoassay, HPLC, and gas chromatography-mass spectrometry methods because of elimination of drug interferences, high throughput, and short chromatographic run time.     

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 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.     

Direct measurement of serum free testosterone by ultrafiltration followed by liquid chromatography tandem mass spectrometry

BackgroundCurrently there is no reliable method suitable for routine measurement of serum free testosterone (FT).AimTo develop such a method involving liquid chromatography tandem mass spectrometry (LC-IDMS/MS) that directly detects and quantifies the FT present in serum.MethodsUltrafiltrate testosterone obtained from 0.5 mL of serum was partially purified by liquid/liquid extraction and quantified using an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source. Using split samples serum free testosterone was compared between direct ultrafiltration (UF) coupled LC-MS/MS, analogue FT immunoassay, free testosterone calculated from mass action equations (cFT) and with equilibrium dialysis (ED) coupled LC-MS/MS.ResultsTotal imprecision determined over twenty runs was < 6% at 67 pmol/L and 158 pmol/L FT. The dynamic response was linear up to at least 2500 pmol/L while physical LLOQ (18 % CV) equaled 16 pmol/L. The UF method agreed poorly with analogue immunoassay (correlation coefficient 0.667; bias ? 81%), somewhat better against cFT when total testosterone was determined by immunoassay (correlation coefficient 0.816, bias 21% ) and still better yet against cFT when total testosterone was determined by LC-MS/MS (correlation coefficient 0.8996, bias 10%). Agreement was closest with ED method (correlation coefficient 0.9779, bias 2.4%).ConclusionWe present a relatively simple UF coupled LC-MS/MS definitive method that measures serum free testosterone. The method is relatively fast, reliable and is suitable for the routine clinical laboratory practice.     

Rapid determination of serum testosterone by liquid chromatography-isotope dilution tandem mass spectrometry and a split sample comparison with three automated immunoassays

Objectives

To develop a rapid convenient-to-implement high performance liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) method for determination of serum testosterone concentration in routine clinical laboratories.

Methods

Following extraction by organic solvents, an Agilent 1200 Series HPLC system coupled to an API 5000 mass spectrometer equipped with an atmospheric pressure chemical ionization ion source was used to separate, detect and quantify serum testosterone. Ion-transitions of m/z 289.2 → 109.1 and 294.2 → 113.2 were used to monitor testosterone and testosterone-2,2,4,6,6-d₅, respectively.

Results

Functional sensitivity was 0.056 nmol/L (CV 20%). Within-run and total imprecision were 4.6% and 5.2% at 1.3 nmol/L, 2.4% and 4.3% at 11.0 nmol/L, and 1.9% and 1.9% at 23.4 nmol/L respectively. The LC-MS/MS method agreed closely with three automated immunoassays when the concentration of testosterone exceeded 3 nmol/L. However, the immunoassays showed a positive bias at concentrations below 3 nmol/L.

Conclusion

This method provides a rapid, simple, highly selective and sensitive procedure that can be easily used for determination of serum testosterone in routine clinical laboratories. It measures serum testosterone precisely and accurately at concentrations found in children and adults of both genders.     

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.     

Analysis of methylmalonic acid in plasma by liquid chromatography-tandem mass spectrometry

BACKGROUND: Methylmalonic acid (MMA) is a biochemical marker for cobalamin deficiency, particularly in cases where the cobalamin concentration is moderately decreased or in the low-normal range. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is a rapid, robust method that has been used in MMA analysis. We developed a simple method combining solid-phase extraction (SPE) and derivatization to prepare serum or plasma for LC-MS/MS analysis of MMA. METHODS: Deuterated internal standard d(3)-MMA was added to serum or plasma before SPE on strong anion-exchange (SAX) columns. After elution with HCl-butanol (10:90 by volume) and addition of 1 g/L formic acid, the samples were simultaneously derivatized and evaporated by heating to 70 degrees C for 15 min followed by 54 degrees C overnight in uncapped vials. Acetonitrile and 1 g/L formic acid were added to the samples before injection into the LC-MS/MS system. MMA and d(3)-MMA were quantified in the multiple-reaction monitoring mode. Calibrators were prepared in serum by the standard addition method. RESULTS: The MMA assay was linear up to 200 micromol/L. Interassay CVs were 6.7%, 5.0%, and 5.0% for mean concentrations of 0.15, 0.36, and 0.65 micromol/L, respectively. CONCLUSIONS: Our simplified sample preparation and derivatization method is suitable for use in MMA analyses. MMA elutes with the derivatization reagent, and derivatization and evaporation are performed simply by leaving the uncapped vials in a heating block overnight. The method shows good linearity and precision.     

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.     

Methylmalonic acid measured in plasma and urine by stable-isotope dilution and electrospray tandem mass spectrometry

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B(12) deficiency. METHODS: We added 1 nmol of the internal standard MMA-d(3) to 500 microL of plasma or 100 microL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 degrees C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d(3,) respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. RESULTS: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 micromol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030 x -0.032 (S(y|x) = 1.03 micromol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3. 8-8.5% and 1.3-3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 micromol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 micromol/L), 96.0% (0.60 micromol/L), and 94.8% (2.02 micromol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100-150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. CONCLUSIONS: This method is well suited for large-scale MMA testing (> or =100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated.     

Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3

BACKGROUND: Measurement of 25-hydroxyvitamin D2 and D3 (25-OH D2 and D3) is essential for investigating vitamin D deficiency. Competitive binding techniques are unable to distinguish between the 2 metabolites and suffer from interference from other hydroxy metabolites of vitamin D. METHODS: We used isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for routine determination of 25-OH D2 and D3 with a stable-isotope-labeled internal standard (IS). Serum samples (100 microL) were denatured with methanol-propanol containing IS, vortex-mixed, extracted into hexane, and dried under nitrogen. The reconstituted extract was chromatographed on a BDS C8 HPLC column, and the metabolites and IS were detected by electrospray ionization MS/MS in multiple-reaction monitoring mode. RESULTS: 25-OH D2 and D3 and the IS nearly coeluted, whereas 1alpha-hydroxyvitamin D3 was separated; total run time was 8 min. The interassay CVs for 25-OH D2 were 9.5% and 8.4% at 52 and 76 nmol/L, respectively, and for 25-OH D3 were 5.1% and 5.6% at 55 and 87 nmol/L, respectively. The detection limit of the present method was <4 nmol/L for both metabolites. Method comparison with a commercial RIA measuring total 25-hydroxyvitamin D showed good correlation: y=0.97x - 2.7 nmol/L (r=0.91). The analytical system can assay 100 samples in 12.5 h. CONCLUSIONS: This simple robust interference-free LC-MS/MS assay is suitable for routine measurement of the 25-hydroxy metabolites of vitamins D2 and D3 in human serum. The assay has been in use for 9 months and has been used to assay more than 6000 routine samples.     

Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay

BACKGROUND: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum. METHODS: We quantitatively isolated folates from 275 microL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C(8) analytical column. (13)C-labeled folates were used as internal standards. RESULTS: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were approximately 9% lower than results obtained with LC/MS/MS. The microbiologic assay gave approximately 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5-75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF. CONCLUSIONS: This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.     

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.     

Measurement of late-night salivary cortisol with an automated immunoassay system.

BACKGROUND: Measurement of late-night salivary cortisol concentrations is increasingly used as a screening test in suspected Cushing's syndrome. Cortisol concentrations are typically extremely low in late-night samples and discordant assay-specific reference ranges have been reported. Therefore, the aim of our study was to assess the analytical performance of the first automated cortisol immunoassay specified for salivary measurements and to establish late-night sampling reference-range data for this test. METHODS: Salivary cortisol was measured using the Roche Cobas Cortisol assay (Roche Diagnostics). Five salivary pools in different concentration ranges were used to assess the inter-assay imprecision of this test in a two-centre evaluation protocol including two reagent lots. Linearity was tested by serial dilution. Salivary samples were obtained at 23:00 h from 100 apparently healthy volunteers using a commercially available salivary sampling device (Salivette, Sarstedt). A subset of 20 samples was used for method comparison with isotope dilution liquid chromatography-tandem mass spectrometry. RESULTS: Inter-assay coefficients of variation (n=20) between 11.6% and 40.4% were found for mean cortisol concentrations between 12.9 and 2.6 nmol/L, with an estimated functional sensitivity of approximately 5.0 nmol/L. The test also gave linear results in the lowest concentration range between 1.0 and 8.3 nmol/L. Mean late-night salivary cortisol of 5.0 nmol/L was found for healthy individuals; the absolute range was 1.4-16.7 nmol/L, and the 95th percentile was 8.9 nmol/L. Substantially lower concentrations were found with isotope dilution LC-MS/MS compared to immunoassay results (mean concentrations 1.8 and 4.4 nmol/L, respectively). CONCLUSIONS: The automated assay investigated was found to offer acceptable analytical performance in the very low concentration range required for late-night salivary cortisol, despite a very short turn-around time. Using this assay, late-night salivary cortisol concentrations below 8.9 nmol/L are typically found in healthy volunteers.