Pediatric reference intervals for free thyroxine and free triiodothyronine.

BACKGROUND: The clinical value of free thyroxine (FT(4)) and free triiodothyronine (FT(3)) analysis depends on the reference intervals with which they are compared. We determined age- and sex-specific reference intervals for neonates, infants, and children 0-18 years of age for FT(4) and FT(3) using tandem mass spectrometry. METHODS: Reference intervals were calculated for serum FT(4) (n = 1426) and FT(3) (n = 1107) obtained from healthy children between January 1, 2008, and June 30, 2008, from Children's National Medical Center and Georgetown University Medical Center Bioanalytical Core Laboratory, Washington, DC. Serum samples were analyzed using isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with deuterium-labeled internal standards. RESULTS: FT(4) reference intervals were very similar for males and females of all ages and ranged between 1.3 and 2.4 ng/dL for children 1 to 18 years old. FT(4) reference intervals for 1- to 12-month-old infants were 1.3-2.8 ng/dL. These 2.5 to 97.5 percentile intervals were much tighter than reference intervals obtained using immunoassay platforms 0.48-2.78 ng/dL for males and 0.85-2.09 ng/dL for females. Similarly, FT(3) intervals were consistent and similar for males and females and for all ages, ranging between 1.5 pg/mL and approximately 6.0 pg/mL for children 1 month of age to 18 years old. CONCLUSIONS: This is the first study to provide pediatric reference intervals of FT(4) and FT(3) for children from birth to 18 years of age using LC/MS/MS. Analysis using LC/MS/MS provides more specific quantification of thyroid hormones. A comparison of the ultrafiltration tandem mass spectrometric method with equilibrium dialysis showed very good correlation.

Rapid measurement of estrogens and their metabolites in human serum by liquid chromatography-tandem mass spectrometry without derivatization.

OBJECTIVES: The steroids estradiol (E2), estrone (E1), and estriol (E3) are the major estrogens. E1/E2 and their metabolite 16-hydroxyestrone (16-OHE1, known to be carcinogenic) could be involved in the development of many cancers including human breast cancer. The aim of the current study was to develop a rapid and simple high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to simultaneously measure E1, E2, E3 and 16-OHE1 in human serum without the need for solid phase extraction or derivatization. METHODS: An API-5000 triple-quadrupole mass spectrometer coupled with electrospray ionization (ESI) source and Shimadzu HPLC system was used employing isotope dilution with deuterium-labeled internal standard (IS) for each analyte. Quantitation by multiple reaction monitoring (MRM) analysis was performed in negative ion mode. RESULTS: The limits of detection were 1.0 pg/mL for E1 and 16-OHE1 and 2.0 pg/mL for E2 and E3. Within-day CVs were <6.5% for all analytes tested and between-day CVs ranged from 4.5% to 9.5%. Recovery ranged from 88% to 108%. CONCLUSION: This method allows for the simultaneous measurement of four estrogens in human serum within 8 min. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing, micro sample requirement, and high throughput.

Determination of levetiracetam in human plasma/serum/saliva by liquid chromatography-electrospray tandem mass spectrometry.

BACKGROUND: Levetiracetam (Keppra) is a novel antiepileptic drug recently approved by the U.S. Food and Drug Administration as an add-on therapy in the treatment of partial onset seizures in patients. We developed and describe a simple and rapid high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) assay for the determination of levetiracetam in human matrix (plasma, serum, or saliva). METHODS: An API-3000 or API-4000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the IonSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing ritonavir as internal standard (IS) for levetiracetam. One hundred microliters of serum (or plasma, saliva) was deproteinized by adding 150 microl of acetonitrile containing internal standard. After centrifugation, 100 microl of supernatant was diluted with 300 microl of water and 10 microl aliquot was injected onto a C-18 column. After a 2.5 min wash the valve was activated to initiate the isocratic elution program which eluted the levetiracetam and internal standard into the MS/MS system. Quantitation by MRM analysis was performed in the positive ion mode. Within-day and between-day imprecision were evaluated for levetiracetam using three levels of in-house controls. Reliability and accuracy of this method were assessed by comparison of targets with external QC material (ChromSystems), between laboratory comparisons and by recovery studies. RESULTS: Within-day coefficients of variation (CVs) were <6.1% and between-day CVs were <8.2%. The average spiked recoveries of levetiracetam added to the drug-free human plasma samples were 108% at low concentration level and 103% at high concentration level. CONCLUSIONS: The method was found both specific and sensitive for the rapid and accurate measurement of levetiracetam in human matrices and correlated well with the Quest/Chantilly tandem mass spectrometric method (r=0.983).

Use of steroid profiles in determining the cause of adrenal insufficiency.

HYPOTHESIS: A cortisol response to adrenocorticotropin injection is the standard test for diagnosing adrenal insufficiency. Multiple steroid hormones can now be accurately measured by tandem mass spectrometry in a single sample. The study objective was to determine whether a steroid profile, created by simultaneous measurement of 10 steroid hormones by tandem mass spectrometry, would help determine the cause of adrenal insufficiency. DESIGN: A 10-steroid profile was measured by tandem mass spectrometry during the performance of a standard high dose cortrosyn stimulation test. The steroids were measured at baseline, 30, and 60min following synthetic adrenocorticotropin injection. Adrenal insufficiency was defined as a peak cortisol level of less than 20microg/dL. Testing was conducted in the general clinical research center of a university medical center. Normal volunteers, patients suspected of having adrenal insufficiency, and patients with known adrenal insufficiency participated. RESULTS: Our results showed that adrenal insufficiency of any cause was adequately diagnosed using the response of 11-deoxycortisol, dehydroepiandrosterone, or these analytes combined in a two-steroid profile. A three-steroid profile yielded a test with 100% accuracy for discriminating primary adrenal insufficiency from normal status. Primary adrenal insufficiency was well separated from secondary adrenal insufficiency using only a single aldosterone value. 11-Deoxycortisol, dehydroepiandrosterone, and a two-steroid profile each provided fair discrimination between secondary adrenal insufficiency and normal status. CONCLUSIONS: We conclude that stimulated levels of aldosterone, 11-deoxycortisol, dehydroepiandrosterone, and a two- or three-steroid profile provided additional discrimination between states of adrenal sufficiency and insufficiency. It is proposed that a steroid profile measuring cortisol, aldosterone, 11-deoxycortisol, and dehydroepiandrosterone would potentially improve the ability to determine the cause of adrenal insufficiency.

Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry.

BACKGROUND: Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandem MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 ul serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 microl. METHODS: An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 microl of acetonitrile containing internal standards. After centrifugation, 450 microl of supernatant were diluted with 900 microl of water and 1000 microl aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study. RESULTS: Within-day CVs were <11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%. CONCLUSIONS: This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput.

Steroid hormone levels in pregnancy and 1 year postpartum using isotope dilution tandem mass spectrometry.

OBJECTIVE: To establish normal, trimester-specific reference intervals for serum 17beta-estradiol, progesterone (P), 17alpha-hydroxyprogesterone, cortisol, 11-deoxycortisol, androstenedione, DHEA, and DHEAS, measured simultaneously using isotope dilution tandem mass spectrometry. DESIGN: Sequential cohort study. PATIENT(S): Healthy women undergoing a normal pregnancy (age, 25-38 years; mean, 30 years) attending a prenatal well clinic at gestation weeks 12, 22, and 32 and approximately 1 year postpartum. MAIN OUTCOME MEASURE(S): Trimester-specific reference intervals of endogenous steroid hormones analyzed using an isotope dilution tandem mass spectrometer equipped with an atmospheric pressure photoionization source with deuterium-labeled internal standards. RESULT(S): Serum estradiol, P, 17alpha-hydroxyprogesterone, and 11-deoxycortisol increased throughout pregnancy; cortisol increased up to the second trimester and then remained steady, while androstenedione increased by 80 percent by gestation week 12, then remained constant. Serum DHEA-S decreased by 50% by the third trimester. CONCLUSION(S): Trimester-specific reference intervals are reported for eight serum steroids. The ratios of individual serum hormone concentrations during pregnancy relative to their 1-year postpartum concentrations illustrate the expected normal trends of changes in hormone concentrations during pregnancy.

Rapid measurement of plasma acylcarnitines by liquid chromatography-tandem mass spectrometry without derivatization.

BACKGROUND: Tandem mass spectrometry (MS/MS) is being increasingly used to identify and measure acylcarnitines in blood and urine of children suspected of having fatty oxidation disorders and other inborn errors of metabolism. Rapid MS/MS analysis requires simple and efficient sample preparation. We developed a LC-MS/MS method for the online extraction of acylcarnitines in plasma without derivatization that requires only precipitation of proteins by acetonitrile followed by centrifugation, thus increasing efficiency. METHODS: An API-3000 tandem mass spectrometer (SCIEX, Toronto, Canada) equipped with electrospray ionization (ESI), TurboIon Spray source, three Shimadzu LC10AD micropumps and autosampler (Shimadzu Scientific Instruments, Columbia, MD) was used to perform the analysis. Within-day and between-day imprecision was evaluated for 10 analytes in the MRM mode using 3 levels of controls. Accuracy was determined by comparing the method with another MS/MS procedure and by recovery experiments. Sensitivity and specificity were evaluated by identifying patient samples under a wide variety of clinical conditions. RESULTS: Within-day CVs was <10% for all analytes tested and between-day CVs ranged from 4.4% to 14.2%. The method was linear in the range between 1.0 and 100 micromol/l for C2 and 0.1 and 10 micromol/l for the other acylcarnitines. The results of the comparison study yielded r values ranging between 0.948 and 0.999. Recovery ranged from 84% to 112%. The method correctly identified patients with a variety of fatty acid oxidation disorders and organic acidemias. CONCLUSIONS: Our method is a simple procedure for the analysis of acylcarnitines in plasma with minimal sample preparation. It is thus ideal in a routine clinical setting where efficient processing of clinical samples is necessary to reduce turnaround time under conditions of high-throughput.

Steroid hormones: relevance and measurement in the clinical laboratory.

Steroid hormones are synthesized in the adrenal cortex, the gonads, and the placenta; are all derived from cholesterol and many are of clinical importance. This article addresses the relevance and methods of measurement of steroid hormones in the clinical laboratory.