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Liquid chromatography tandem mass spectrometry is one of the most specific techniques available in clinical laboratories. In the past, immunoassays were the primary methodology for analysis of steroids in biological samples because they are rapid and easy to perform. However, these methods were shown to suffer from the lack of specificity for measuring many of the diagnostically important steroids. LC-MS/MS overcomes many of the limitations of immunoassays, enhances diagnostic utility of the testing, and expands diagnostic capabilities in endocrinology. In addition to the superior quality of the measurements, LC-MS/MS allows high throughput testing using small sample volume with minimal sample preparation, and frees the laboratory from dependence on suppliers of assay specific reagents. LC-MS/MS is being widely employed for routine measurement of steroids, and the methodology plays an important role in the standardization and harmonization of measurements among clinical laboratories.

Background: Measurements of free thyroxine (FT4) and free triiodothyronine (FT3) are important for the diagnosis and monitoring of thyroid diseases. Considerable differences among methods limit their clinical utility, however, and accurate methods are needed for various clinical specimens. We describe a direct equilibrium dialysis (ED)–liquid chromatography (LC)/tandem mass spectrometry (MS/MS) method for FT4 and FT3.Methods: ED was selected as the separation step. Serum samples were dialyzed 1:1 against a simple protein-free buffer for 20 h at 37 °C. Thyroid hormones in dialysates were purified by online solid-phase extraction (SPE), then chromatographically separated and quantified in positive ion and multiple reaction monitoring modes.Results: For FT4 and FT3, the lower and upper limits of quantification were 1 ng/L (pg/mL) and 400 ng/L with total imprecision Conclusions: We developed a simple protein-free buffer and ED procedure. The performance characteristics and high throughput of the LC-MS/MS method with online SPE for FT4 and FT3 (also reverse T3) are sufficient for the intended clinical use.

High-sensitivity measurement of serum estrogens is important in adult and pediatric endocrinology and oncology. We developed a high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of estrone (E1) and estradiol (E2). Aliquots of 200 muL of serum were spiked with internal standard, extracted, derivatized with dansyl chloride, and analyzed by LC-MS/MS using 2-dimensional chromatographic separation. Total imprecision for the method was less than 11%; the limit of quantitation was 1 pg/mL. Reference intervals were established with samples from more than 900 healthy postmenopausal women, men, girls, and boys. Concentrations of estrogens in children reached adult levels by Tanner stage 3. In men and postmenopausal women, the median concentrations of total estrogens (E1 + E2) were 39 and 22 pg/mL, and the median E2/E1 ratios were 0.98 and 0.55, respectively. The method requires a small sample volume and has adequate sensitivity and specificity for analyzing estrogens in samples from postmenopausal women, men, and children.

A new electron ionization source was developed for orthogonal acceleration time-of-flight mass spectrometry (TOFMS) based on the superimposition of a magnetic field around a radio frequency-only (rf-only) ion guide. The cylindrically symmetric magnetic field compresses the electron beam from the electron source into a long narrow volume along the ion guide axis. The magnetic field also helps to maintain a narrow energy distribution of electrons that penetrate the full length of the ion guide despite the influence of the radial rf field. Ionization occurs inside the ion guide with improved efficiency resulting from efficient use of electrons, prolonged interaction time, and nontraditionally large ionization volume. At the same time, the rf field effectively focuses ions radially and confines them to the axis of the ion guide by collisional focusing, leading to high ion transmission efficiency. Furthermore, the source can also be operated in a trap-and-pulse mode to improve the ion sampling duty cycle of orthogonal acceleration TOFMS. To validate the design concept of this new ion source, a simple prototype using a single set of cylindrical rods was constructed and retrofitted to an orthogonal acceleration TOFMS. A significant increase in ion signal intensity was observed by operating the source in a pulsed ion extraction mode. Low detection limits (for example, 12 fg for toluene) were determined at 12.5 spectra s(-1) in the full spectrum mode.

An ODS packing material was specially designed for CEC to achieve high efficiency. The size of the particles was approximately 3.2 μm based on scanning electron microscopy, and the average pore size of the particles was 1500 A by mercury intrusion porosimetry measurement. The packing materials were used in sol-gel bonded continuous-bed columns. The performance of columns containing these 3 μm, 1500 A ODS particles was evaluated for CEC and compared to 3 μm, 80 A ODS particles. The electroosmotic velocity (EOF) for the 1500 A ODS material was slightly greater than that for the 80 A ODS material. An efficiency of 410,000 plates m−1 was obtained for unretained thiourea with a 30 cm × 75 μm i.d. 1500 A ODS column, which was more than 2 times higher than the efficiency obtained from the 80 A ODS column.

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60–200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. β-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, β-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system.

Electrospray ionization has grown to be one of the most commonly used ionization techniques for mass spectrometry, and efforts continue to improve its performance. Typically, the sprayer tip must be very close to the entrance orifice of the mass spectrometer in order to maximize the conduction of ions from the sprayer into the mass spectrometer. However, because of space-charge repulsion, most ions never reach the sampling orifice. In this work, an industrial air amplifier, for which the working mechanism is based on venturi and coanda effects, was added between an electrospray ionization source and a time-of-flight mass spectrometer. When a series of reserpine solutions (0.5, 1.0, 5.0, and 10.0 microM) were monitored using mass spectrometry, an over 5-fold increase in m/z 609.3 ion intensity was measured for a separation distance of 14 mm between the electrospray tip and interface capillary inlet, as compared to when the electrospray tip was in its normal position 1 mm in front of the inlet without the amplifier. When a voltage was applied to the air amplifier to further assist in focusing the electrosprayed ions, an approximately 18-fold increase in m/z 609.3 ion intensity was obtained. In addition, a 34-fold reduction in method detection limit was observed.

Two bonded chiral stationary phases (CSPs), 8-aminoquinoline-2-ylmethyl- and 8-aminoquinoline-7-ylmethyl-diaza-18-crown-6-capped [3-(2-O-beta-cyclodextrin)-2-hydroxypropoxy]propylsilyl silica particles (non-porous, 1.5 microm), have been prepared and evaluated using capillary liquid chromatography at high pressures (or = 8000 p.s.i.). High column efficiency (up to 400 000 plates m(-1)) was achieved for chiral separations. These CSPs with two recognition sites, i.e. substituted-diaza-18-crown-6 and beta-cyclodextrin combined with high chromatographic efficiency provide good resolution of a variety of enantiomers and positional isomers in relatively short times under reversed-phase conditions. After inclusion of a Ni (II) ion from the mobile phase, the positively charged crown ether-capped beta-cyclodextrin facilitates specific static, dipolar, and host-guest complexation interactions with solutes.

Capillary columns packed with small diameter particles typically lead to low permeability and long separation times in high-performance liquid chromatography. Ultrahigh pressures (10,000 p.s.i.; 1 p.s.i. is identical with 6,894.76 Pa) can be used to overcome the limitations that small particles impose. Ultrahigh-pressure liquid chromatography (UHPLC) has demonstrated great potential for high-speed and high-efficiency separations. Decreasing the viscosity of the mobile phase by elevating the temperature could additionally reduce the pressure drop and facilitate the use of longer columns or smaller particles to achieve even higher total plate numbers. For this reason, we investigated the use of elevated temperatures in UHPLC. Water-resistant, flexible heater tape covered with insulation was used to provide the desired heat to the column. Polybutadiene-coated 1 microm nonporous zirconia particles were used because of their chemical stability at elevated temperature. A column efficiency as high as 420,000 plates m(-1) was obtained. The effects of temperature and pressure on the separation of parabens were investigated. Separation of five herbicides was completed in 60 s using 26,000 p.s.i. and 90 degrees C.

Nucleosides in human urine have been studied frequently as a possible biomedical marker for cancers, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. A capillary electrophoretic method that can quantitatively analyze urinary normal and modified nucleosides in less than 40 min with a good resolution and sufficient sensitivity has been developed. Twelve kinds of normal and modified nucleosides were determined in urine samples from 25 healthy persons and 25 cancer patients of 14 kinds of cancers. Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached to 100% and above 85% of the members in the predicting set were correctly classified. In addition, the neural network technique was compared with methods of the principal component analysis and the canonical discriminant analysis. The results demonstrate that the predictive ability of the artificial neural network is stronger than the others in this study.

Stable isotope-labeled internal standards are of great utility in providing accurate quantitation in mass spectrometry (MS). An implicit assumption has been that there is no "cross talk" between signals of the internal standard and the target analyte. In some cases, however, naturally occurring isotopes of the analyte do contribute to the signal of the internal standard. This phenomenon becomes more pronounced for isotopically rich compounds, such as those containing sulfur, chlorine, or bromine, higher molecular weight compounds, and those at high analyte/internal standard concentration ratio. This can create nonlinear calibration behavior that may bias quantitative results. Here, we propose the use of a nonlinear but more accurate fitting of data for these situations that incorporates one or two constants determined experimentally for each analyte/internal standard combination and an adjustable calibration parameter. This fitting provides more accurate quantitation in MS-based assays where contributions from analyte to stable labeled internal standard signal exist. It can also correct for the reverse situation where an analyte is present in the internal standard as an impurity. The practical utility of this approach is described, and by using experimental data, the approach is compared to alternative fits.

Background: Measurement of serum androgens is important in adult, geriatric, pediatric endocrinology, and oncology patients. We developed a liquid chromatography—tandem mass spectrometry (LC-MS/MS) assay for simultaneous measurement of androstenedione, dehydroepiandrosterone (DHEA), and testosterone in these patients. Methods: We spiked 200 μL of serum or plasma with isotope-labeled internal standards and performed extraction with methyl t-butyl ether. We then derivatized the extracts with hydroxylamine and analyzed them by LC-MS/MS using a 2-dimensional chromatographic separation with a 3.5-min analysis time. Results: Total imprecision for each analyte was Conclusions: This LC-MS/MS method has the required lower limit of quantification and specificity for analysis of endogenous concentrations of androgens in all groups studied. Reference intervals were established for healthy children and adults.

Measurement of low concentrations of estrogens, encountered in pre-pubertal children, men, and postmenopausal women, is important for numerous clinical applications. We describe a method for high sensitivity analysis of estrogens that uses two-dimensional chromatographic separation and tandem mass spectrometry detection. Aliquots of serum or plasma samples are combined with stable isotope-labeled internal standard and estrogens are extracted with methyl t-butyl ether. The solvent is evaporated, estrogens derivatized to form dansyl derivatives, and the samples are analyzed. Quantitation is performed using triple quadrupole mass spectrometer equipped with electrospray ion source using positive ion mode ionization and multiple reaction monitoring acquisition.

We describe a method for the quantitative analysis of nicotine, cotinine, trans-3'-hydroxy cotinine, nornicotine, and anabasine in urine, serum, and plasma using liquid chromatography-tandem mass spectrometry. A mix of deuterium-labeled internal standards (IS) is added to a specimen aliquot. The aliquot is extracted using mixed-mode solid phase extraction and eluted into an autosampler vial for injection into an LC-MS-MS system. An Atlantis silica column is used for LC separation in hydrophilic interaction mode. Tandem mass spectrometry detection is performed in positive ion mode with electrospray ionization and two multiple reaction monitoring (MRM) transitions monitored for each analyte and IS.

Measurement of low concentrations of estrogens, encountered in pre-pubertal children, men, and postmenopausal women, is important for numerous clinical applications. We describe a method for high sensitivity analysis of estrogens that uses two-dimensional chromatographic separation and tandem mass spectrometry detection. Aliquots of serum or plasma samples are combined with stable isotope-labeled internal standard and estrogens are extracted with methyl t-butyl ether. The solvent is evaporated, estrogens derivatized to form dansyl derivatives, and the samples are analyzed. Quantitation is performed using triple quadrupole mass spectrometer equipped with electrospray ion source using positive ion mode ionization and multiple reaction monitoring acquisition.

We describe a method for the quantitative analysis of nicotine, cotinine, trans-3'-hydroxy cotinine, nornicotine, and anabasine in urine, serum, and plasma using liquid chromatography-tandem mass spectrometry. A mix of deuterium-labeled internal standards (IS) is added to a specimen aliquot. The aliquot is extracted using mixed-mode solid phase extraction and eluted into an autosampler vial for injection into an LC-MS-MS system. An Atlantis silica column is used for LC separation in hydrophilic interaction mode. Tandem mass spectrometry detection is performed in positive ion mode with electrospray ionization and two multiple reaction monitoring (MRM) transitions monitored for each analyte and IS.

Background: Choline is critical for a variety of biological functions and has been investigated as a biomarker for various pathological conditions including acute coronary syndrome. Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to quantify choline in whole blood and plasma in freshly collected samples prepared with ultrafiltration or protein precipitation. We investigated the effects of preanalytical variables including types of anticoagulants and storage temperature and time. Results: We observed no significant differences in whole-blood choline concentration in EDTA-anticoagulated vs heparin-anticoagulated samples: mean (SD) difference 0.9% (3.2%), P = 0.80. For plasma, choline concentrations with heparin in 5 of 12 volunteers were >10% higher than with EDTA, P = 0.01. One freeze-thaw cycle led to significant mean (SD) increases in choline concentrations in heparin whole blood, 19.3% (11.4%), P 0.33). For freshly collected samples stored at ambient temperature, choline concentrations in all types of samples increased with storage time. For EDTA whole blood, EDTA plasma, and heparin plasma, the choline concentration increased for the first 60 min and then stabilized. For heparin whole blood, the choline concentration continued to increase linearly with storage time for >4 h, at which time the choline concentrations were increased by approximately 50%. Conclusions: Sample collection, storage, and sample preparation procedures are critical for clinical measurements of choline in whole blood and plasma.

Measurements of urinary fractionated metanephrines (normetanephrine and metanephrine), the O-methylated metabolites of norepinephrine and epinephrine, provide a sensitive test for diagnosis of pheochromocytoma. We present evidence suggesting inaccurate metanephrine and normetanephrine calibration by US laboratories. Metanephrines are present in urine mainly as sulfate- and glucuronide-conjugated metabolites produced from free metanephrines by the actions of conjugating enzymes (1). An acid hydrolysis step is usually performed to liberate the free metanephrines from the conjugated metabolites. This step minimizes requirements for high analytical sensitivity, simplifying subsequent measurement. Urinary fractionated metanephrines are usually measured by HPLC with electrochemical detection (HPLC-EC). Gas chromatography with mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) are more recent alternatives offering high sample throughput and improved analytical specificity (2)(3). Clinical laboratories in the US participate in proficiency testing programs, primarily aimed at determining agreement in diagnostic test results among laboratories. Identical survey samples are distributed to participating laboratories, with results stratified according to testing methodologies. Laboratories that report results deviating by more than a certain amount from the mean of their peer groups fail the proficiency test. Until the spring of 2004, the only source of commercially available urine metanephrine calibrators in the US was Bio-Rad. As with … aE-mail steve_binder{at}bio-rad.com

Objectives: The necessity of confirmation of compound identity in quantitative analysis is well recognized for methods utilizing single mass spectrometry detection but is not commonly addressed for applications utilizing multiple-stage mass spectrometry (MSn). For MSn detection, no commonly accepted rules for assessment of analytical specificity in quantitative analyses have been established to date. Methods: To assure compound identity, we evaluated approaches based on monitoring multiple mass transitions of a target compound followed by comparison of the branching ratios of the mass transitions. Results: Monitoring multiple mass transitions along with evaluation of the ratio of their relative intensities allows the analyst to distinguish the target analyte from interferences in quantitative analysis. The strategy and the acceptance criteria are compound and method specific and should be established during the method development and validation. Conclusions: The certainty of analyte identity is very important in quantitative analysis using MSn detection; methods to verify analyte identity should be used in all critical applications.