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.

Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Quantification of Steroids Androstenedione, Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone.

Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive disorders due to deficiency of 11- or 21-hydroxylase. The analysis of cortisol, androstenedione, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Analysis of specific steroids is also performed in other disorders such as evaluation of hirsutism or infertility in females and hypogonadism in males. Cortisol is generally analyzed by immunoassays, whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC-MS/MS method is described for the simultaneous quantification of androstenedione, 17-hydroxyprogesterone, DHEA, 11-deoxycortisol and testosterone. The method involves addition of labeled internal standards to serum samples and extraction of steroids in methyl tert-butyl ether. The extract is evaporated under stream of nitrogen, and the residue is reconstituted in methanol and analyzed by LC-MS/MS.

Quantification of 25-Hydroxyvitamin D2 and D3 Using Liquid Chromatography-Tandem Mass Spectrometry.

Vitamin D plays an important role not only in bone health but also in many other body functions. Vitamin D deficiency is very common in the general population. Measurement of blood 25-hydroxyvitamin D is a common practice to evaluate vitamin D deficiency. Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are the most commonly used methods for the measurement of 25-hydroxyvitamin D. Immunoassays suffer from specificity issues and do not distinguish between 25-hydroxyvitamin D2 and D3. Therefore, LC-MS/MS is a preferred method for quantification of 25-hydroxyvitamin. We describe an LC-MS/MS method, which involves protein precipitation and analysis of the extract using atmospheric pressure chemical ionization and multiple reaction monitoring. 25-hydroxyvitamin D3-d6 is used as an internal standard. The method is linear from 1-100 ng/mL for both 25-hydroxyvitamin D2 and D3 and has imprecision of <10%.

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.

Quantification of Dehydroepiandrosterone, 11-Deoxycortisol, 17-Hydroxyprogesterone, and Testosterone by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS).

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders due to enzymatic defects in the biosynthetic pathway of cortisol and/or aldosterone. The analysis of cortisol, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Cortisol is generally analyzed by immunoassays whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC/MS/MS method is described for the simultaneous quantification of 17-hydroxyprogesterone, DHEA, 11-deoxycortisol, and testosterone. Stable-isotope labeled internal standards are added to serum samples and steroids are extracted by liquid-liquid extraction using methyl tert-butyl ether. The extract is evaporated under stream of nitrogen and the residue is reconstituted in methanol and analyzed by LC/MS/MS.

Cetirizine Quantification by High-Performance Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS).

A multiple reaction monitoring (MRM), positive ion electrospray ionization, LC/MS/MS method is described for the quantification of cetirizine. The compound was isolated from human plasma by protein precipitation using acetonitrile. Cetirizine d4 was used as an internal standard. Chromatographic conditions were achieved using a C18 column and a combination of ammonium acetate, water, and methanol as the mobile phase. MRMs were: cetirizine, 389.26 → 165.16, 201.09; cetirizine d4, 393.09 → 165.15, 201.10. Calibration curves were constructed by plotting the peak area ratios of the calibrators' target MRM transition area to labeled internal standard target MRM transition area versus concentration.

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.

Simultaneous determination of cyclosporine, sirolimus, and tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry.

A multiple reaction monitoring, positive ionization electrospray, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous quantification of cyclosporine, sirolimus, and tacrolimus in human whole blood. Proteins in the samples are precipitated with a mixture of methanol and zinc sulfate. The supernatant is injected into the LC-MS/MS for analysis. Chromatography involves the use of a C18 column and ammonium acetate/water/methanol-containing mobile phases. The MS/MS is operated in positive ion electrospray mode. Quantification is achieved by comparing peak area ratios of MRMs of analytes and internal standards with that of calibrators. Calibration curves are constructed from peak area ratios of MRMs of calibrators and internal standards versus concentrations. MRMs used are ascomycin (m/z 809.5 → 756.5), cyclosporine A (m/z 1,219.9 → 1,203.1), cyclosporine D (m/z 1,234.0 → 1,217.0), sirolimus (m/z 931.6 → 864.5), and tacrolimus (m/z 821.5 → 768.4).