Technical Note: Significant Positive Bias in Lower Concentrations but Negative Bias in Higher Concentrations in Testosterone Measurement Using Beckman Access Immunoassay Compared to a Liquid Chromatography-Tandem Mass Spectrometry Reference Method.

OBJECTIVE: Testosterone is the principal male sex hormone and is secreted primarily by the testes. In most clinical laboratories testosterone is routinely measured for diagnosis of male hypogonadism or androgen excess in females using FDA approved immunoassays. We compared testosterone values measured by Beckman access immunoassay with those measured by a reference LC-MS/MS method. METHODS: Testosterone was measured in 36 patients using left over serum or plasma specimens by both Beckman immunoassay on the DXI 800 analyzer and a reference LC-MS/MS method. RESULTS: We observed overall significant negative bias of approximately 31.9 % when testosterone values obtained by the reference LC-MS/MS method were plotted in the x-axis and the corresponding testosterone values using the immunoassay in the y-axis, as regression equation was y=0.6887x+38.81 (n=36). The corresponding Deming regression was y=0.6639x+34.7163. However, in eight specimens with low testosterone concentrations, immunoassays significantly overestimated testosterone concentrations. CONCLUSIONS: Immunoassays may underestimate the true testosterone concentration in males but overestimate in females with low testosterone concentration. Therefore, for diagnosis of hypogonadism in males and androgen excess in females, testosterone values obtained by Beckman Access immunoassay on the DXI 800 analyzer should be interpreted with caution.

Evaluation of a Delta-9-Tetrahydrocannabinol Carboxylic Acid (Δ9-THC-COOH) Immunoassay and a Gas Chromatography-Mass Spectrometry (GC-MS) Method for the Detection of Delta-8-Tetrahydrocannabinol Carboxylic Acid (Δ8-THC-COOH).

BACKGROUND: Delta-8 tetrahydrocannabinol (Δ8-THC) is a naturally occurring or synthetically prepared cannabinoid that elicits psychological and physiological experiences commonly reported for its more infamous isomer, delta-9 tetrahydrocannabinol (Δ9-THC). Unlike Δ9-THC, Δ8-THC products are generally legal under federal law and there has been a rise in their usage. One of the main targets for detection and quantitation of Δ9-THC is its inactive metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH). METHODS: This study evaluated the ability of the currently used Δ9-THC-COOH immunoassay and gas chromatography-mass spectrometry (GC-MS) methods to detect 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THC-COOH) and distinguish it from Δ9-THC-COOH. RESULTS: The EMIT II Plus® Cannabinoid immunoassay for Δ9-THC-COOH with a cutoff of 20 ng/mL showed positive results for Δ8-THC-COOH with concentrations of 30 ng/mL or higher. Although many of the ion fragments generated by mass spectrometry were found to overlap between the 2 compounds, the GC-MS method presently used to quantify Δ9-THC-COOH separated the 2 compounds sufficiently to identify them independently by relative retention time. CONCLUSION: Current immunoassays and GC-MS methods should be evaluated for the ability to detect and distinguish the presence of Δ8-THC-COOH.

Quantitation of Mycophenolic Acid and Mycophenolic Acid Glucuronide in Serum or Plasma by LC-MS/MS.

Mycophenolic acid (MPA) is an immunosuppressant that is used as an adjunct therapy in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, mass spectrometry methods are preferred due to their higher specificity. Herein, we describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of MPA levels and its conjugate, MPA glucuronide (MPAG). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a zinc sulfate solution and acetonitrile containing deuterated internal standards (MPA-d3 and MPAG-d3). The resulting protein-free supernatant is injected into the LC-MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitation of mycophenolic acid and mycophenolic acid glucuronide in serum or plasma by LC-MS/MS.

Determination of Mycophenolic Acid and Mycophenolic Acid Glucuronide Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS).

Mycophenolic acid (MPA) is an immunosuppressant that is used in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring of MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, due to their higher specificity, mass spectrometry methods are preferred. In this unit, we describe a liquid chromatography tandem mass spectrometry (LC/MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of levels of MPA and its conjugate MPA glucuronide (MPAG). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a solution containing zinc sulfate and acetonitrile that has been spiked with deuterated internal standards. The resulting protein-free supernatant is injected into the LC/MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2018 by John Wiley & Sons, Inc.

A simple isotope dilution electrospray ionization tandem mass spectrometry method for the determination of free phenytoin.

BACKGROUND: Phenytoin (diphenylhydantoin) is an anticonvulsant drug frequently prescribed for the treatment of many types of seizures. Because the drug is highly protein bound (90%-95%) and many conditions can displace the drug from proteins, the measurement of free phenytoin is warranted. Due to the unavailability of free phenytoin assays in many chemistry analyzers or limitations of immunoassays, chromatographic methods such as liquid chromatography-tandem mass spectrometry (LC-MS-MS) are preferred for the assay of free phenytoin. METHODS: The sample preparation involved ultrafiltration of serum or plasma to separate free phenytoin. Acetonitrile containing internal standard, phenytoin-d10, was added to the ultrafiltrate. The samples were centrifuged, and supernatants were injected into an LC-MS-MS involving reverse phase Ultra BiPh 5-µm × 50 × 2.1-mm analytical column, and mobile phases, water and methanol containing 0.1% formic acid. The mass/charge (m/z) transitions were as follows: phenytoin -253.0 > 182.2 and 253.0 > 104.00; phenytoin-d10 -263.2 > 192.12. RESULTS: Linearity of the method ranged from 0.1 to 4.0 µg/mL. Within-run and between-run imprecision values were <5% and <10%, respectively. The samples were stable for 2 weeks at 4°C and 4 weeks at -20°C. The method compared well with the laborious liquid-liquid extraction method and did not show any significant ion suppression or enhancement. CONCLUSIONS: A simple LC-MS-MS method was developed for the assay of free phenytoin. The method does not require laborious liquid-liquid or solid-phase extraction. The method has high analytical sensitivity, low imprecision, and a wide analytical measurement range.

A simple, rapid atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry method for the determination of 25-hydroxyvitamin D2 and D3.

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods.