Quantification of mycophenolic acid and glucuronide metabolite in human serum by HPLC-tandem mass spectrometry

BACKGROUND: The potent immunosuppressant mycophenolic acid (MPA) is metabolized to an inactive glucuronide (MPAG). The extent of metabolism varies among individuals, and the MPAG formed can be hydrolyzed to MPA and can displace MPA from serum albumin, creating a potential need to monitor both MPA and MPAG. METHODS: After addition of the carboxybutoxy ether of MPA (MPAC) as internal standard, MPA and MPAG were isolated from serum by acidification followed by solid-phase extraction. Gradient chromatographic separation was performed on a Waters Atlantis reversed-phase liquid chromatography (HPLC) column, and the compounds were quantified by electrospray ionization tandem mass spectrometry (MS/MS) in the multiple-reaction monitoring mode. Results obtained by HPLC-MS/MS were compared with an HPLC assay using ultraviolet detection (HPLC-UV) performed at a reference laboratory. RESULTS: MPAG, MPA, and MPAC were fully separated during a 7.0-min run time. Precision at both low and high concentrations of MPA ad MPAG met the suggested method validation criteria from a consensus panel report on MPA. The extraction efficiencies were 99% for MPA and MPAG. The assay was linear to 16 mg/L for MPA and 200 mg/L for MPAG. Limits of quantification were 0.1 mg/L for MPA and 1 mg/L for MPAG. Regression analysis gave the following results: HPLC-MS/MS = 1.03(HPLC-UV) - 0.03 mg/L (R2 = 0.982) for MPA; and HPLC-MS/MS = 0.93(HPLC-UV) + 0.89 mg/L (R2 = 0.967) for MPAG. CONCLUSION: This HPLC-MS/MS assay can be used to reproducibly quantify MPA and MPAG across a large analytical range in serum from organ transplant patients.     

UPLC MS/MS method for quantification of mycophenolic acid and metabolites in human plasma: Application to pharmacokinetic study

BackgroundWe described the development and full validation of a rapid, high throughput sensible and accurate UPLC method using tandem mass spectrometry detection for mycophenolate acid (MPA) and its metabolites, MPA glucuronide (MPAG) and acyl MPA glucuronide (AcMPAG) concentration determination with MPA-D3 as internal standard in human plasma.MethodsPlasma pretreatment involved a one-step protein precipitation with acetonitrile. The separation was performed by reverse-phase chromatography on a Waters BEH HSST3 100 mm*2.1 mm*1.8 μm column. The multiple reaction monitoring transitions used for quantification were m/z 321.04 → 303.02 for MPA, 524.09 → 303.02 for AcMPAG and MPAG and 324.03 → 306.04 for MPA-D3 in the electrospray positive ionization mode.ResultsThe method was linear over the concentration range of 0.1–20 mg/L for MPA and AcMPAG and 1–200 mg/L for MPAG respectively. The intra- and inter-day precision values were below 14% and accuracy was from 94.0 to 103.3% at all quality control levels. The lower LOQ was 0.1 mg/L for MPA and AcMPAG, 1 mg/L for MPAG.ConclusionSample analysis time was reduced to 7 min including sample preparation. The present method was successfully applied to a pharmacokinetic study following oral administration of enterocoated sodium mycophenolate in de novo renal transplantation.     

Inclusion of MPA and in a rapid multi-drug LC-tandem mass spectrometric method for simultaneous determination of immunosuppressants

BACKGROUND: [corrected] Mycophenolic Acid (MPA) is often co-prescribed as part of a multiple immunosuppressant drug regimen. In this study an established LC-MS/MS method for the measurement of immunosuppressants cyclosporine A, tacrolimus, sirolimus and everolimus was optimized to include MPA without changing the sample pre-treatment and the LC-MS/MS configuration. METHODS: The sample pretreatment for EDTA-plasma was used as for whole blood. After protein precipitation of 50 mul EDTA-plasma fast on-line matrix clean-up was performed using a column switching program. The chromatographic step was optimized to separate MPA and its glucuronide metabolite (MPAG). Multiple reaction monitoring (MRM) was used for detection of MPA (337.7>207.2) and MPAG (513.6>207.2). RESULTS: A total analysis time of 5 min was needed to separate MPA and MPAG. The method was linear between 0.05 and 50 mg/L for MPA. Analytical recoveries were >95%. Variation coefficients ranged between 3.1 and 4.1%. Method comparison for MPA was performed using a commercial HPLC-UV test. The Pearson correlation coefficients were >0.9. The Bland-Altman plot showed an excellent agreement between LC-MS/MS and HPLC-UV quantification. CONCLUSION: We present a robust online SPE-LC-MS/MS platform for a simultaneous and fast daily therapeutic drug monitoring of five immunosuppressive drugs in whole blood and plasma samples.     

Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit

BACKGROUND: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. METHODS: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. RESULTS: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. CONCLUSIONS: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.     

Stability of mycophenolic acid and glucuronide metabolites in human plasma and the impact of deproteinization methodology

BACKGROUND: In recent years there is growing interest in therapeutic drug monitoring of mycophenolic acid (MPA) and its glucuronide metabolites MPAG and AcMPAG. Like other acyl glucuronide metabolites, AcMPAG has a limited stability, but this aspect has received little attention. METHODS: Plasma sample deproteinization with perchloric acid 2 M (method A) was compared to metaphosphoric acid 15% (method B). Stability of MPA, MPAG and AcMPAG in acidified and non-acidified plasma stored at room temperature, 4 degrees C, -20 degrees C and -80 degrees C was assessed over short and long time intervals using HPLC-UV methodology. RESULTS: The area ratio of AcMPAG/IS on spiked plasma at pH 2.5 with method A was 63% of the respective ratio in water, in contrast to 102% with method B, suggesting partial deconjugation and/or incomplete release of AcMPAG from proteins with method A. At room temperature, AcMPAG concentrations in both whole blood and non-acidified plasma decreased significantly after 2-5 h. MPA, MPAG and AcMPAG concentrations remained stable in acidified plasma stored at -20 degrees C and -80 degrees C, but not longer than 5 months after collection. CONCLUSIONS: It is concluded that adequate sample collection, storage measures and deproteinization methods should be applied in order to avoid deconjugation and hence underestimation of MPA, MPAG and AcMPAG concentrations.     

Measurement of mycophenolic acid and its glucuronide using a novel rapid liquid chromatography-electrospray ionization tandem mass spectrometry assay

ObjectivesMycophenolic acid (MPA), the active metabolite of the ester prodrug mycophenolate mofetil is an immunosuppressant which selectively inhibits inosine-monophosphate dehydrogenase. The requirement for therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and also its metabolite mycophenolic acid glucuronide (MPAG).Design and methodsWe developed a robust, rapid, sensitive and highly specific HPLC–electrospray ionization mass spectrometry method to assay MPA and its metabolite MPAG in human plasma and serum. Ion suppression was investigated by a post column infusion experiment.ResultsDetermination of MPA and MPAG were performed during a 3.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05 to 100 mg/L (r > 0.999) and 6 to 400 mg/L r > 0.998, respectively. Limits of detection were 0.009 mg/L for MPA and 4.5 mg/L for MPAG and lower limits of quantification were 0.011 mg/L for MPA and 4.9 mg/L for MPAG.Interassay imprecision was < 6.0% for both substances. Mean recovery was 48.9% (range 43.3–60.0%) for MPA and 112.2% (range 95.0–127.0%) for MPAG. Agreement was relatively good for MPA (n = 122) between the presented method and a validated ELISA method (Viva analyzer, Siemens Medicals Solutions Diagnostics, NY). The Passing–Bablok regression line was: EMIT = 0.91 (LC–MS/MS) + 0.17 [mg/L]; r = 0.97.ConclusionsThis simple, robust and interference-free LC–MS/MS assay allows the rapid and accurate determination of MPA and MPAG in human plasma and other body fluids.     

Cyclosporine concentration-dependent increase in concentration ratio of mycophenolic acid acyl and phenol glucuronides to mycophenolic acid in stable kidney transplant recipients

ObjectivesThe aim of this study was to evaluate the influence of cyclosporine (CyA) and tacrolimus (Tac) on the pharmacokinetics of mycophenolic acid (MPA) and its glucuronides.Design and methodsKidney transplant recipients treated with mycophenolate mofetil and CyA (n = 18) or Tac (n = 17) in the stable phase were enrolled. The dependence of the trough concentration (C₀) ratios of MPA acyl glucuronide (AcMPAG) to MPA (AcMPAG/MPA) and MPA phenol glucuronide (MPAG) to MPA (MPAG/MPA) on CyA C₀ or Tac C₀ was evaluated.ResultsAcMPAG C₀ and MPAG C₀ were significantly higher in CyA- than Tac-treated recipients (P = 0.04 and 0.02, respectively). AcMPAG/MPA and MPAG/MPA were significantly correlated to CyA C₀ (r = 0.75, P < 0.01 and r = 0.81, P < 0.01, respectively), but not to Tac C₀.ConclusionsCyA increased AcMPAG/MPA as well as MPAG/MPA in a concentration-dependent manner, suggesting that higher CyA may cause AcMPAG-related adverse reactions. Tac did not alter pharmacokinetics of MPA and its glucuronides.     

The acyl glucuronide metabolite of mycophenolic acid induces tubulin polymerization in vitro

ObjectivesThe acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) forms covalent protein adducts and possesses antiproliferative properties independent of IMPDH inhibition. The underlying mechanism is unknown. Disorganized tubulin polymerization prevents cell cycle progression. We investigated whether AcMPAG interacts with tubulin polymerization.Design and methodsAcMPAG (1.0–100 μM) was incubated with bovine tubulin in the presence of GTP. Polymerization was followed at 340 nm. The time until onset and the extent of polymerization were determined. MPA (100 μM), phenolic glucuronide MPAG (100 μM), and paclitaxel (10 μM) served as controls.ResultsMPAG was without effect. The AcMPAG effect on tubulin polymerization was dose dependent and significantly stronger (about 2.5-fold) than that of MPA (n =   4; p <   0.05), but weaker than paclitaxel.ConclusionsMPA and AcMPAG can induce tubulin polymerization in the presence of GTP with AcMPAG being significantly stronger. This property of AcMPAG may contribute to its IMPDH independent antiproliferative effect.     

Rifampin induces alterations in mycophenolic acid glucuronidation and elimination: implications for drug exposure in renal allograft recipients

BACKGROUND: Exposure to mycophenolic acid (MPA) and its main metabolites (MPA 7-O-glucuronide [MPAG] and MPA acyl-glucuronide [AcMPAG]) is characterized by a large interindividual and intraindividual variability, resulting in part from variability in glucuronidation (via uridine diphosphate-glucuronosyltransferase isoforms) and excretion via multidrug resistance-associated protein 2 (MRP2). It can be hypothesized that drugs interfering with glucuronidation and excretion will alter (Ac)MPA(G) exposure. METHODS: This prospective, open-label, nonrandomized, controlled pharmacokinetic interaction study included 8 stable renal allograft recipients, all treated with mycophenolate mofetil. Rifampin (INN, rifampicin), administered once daily (600 mg/d) for 8 days, was used as the probe drug because of its known effects on both uridine diphosphate-glucuronosyltransferase activity and MRP2 transport capacity. A 12-hour pharmacokinetic time-concentration profile was assessed before rifampin administration was started, and this was repeated on the last day of rifampin administration. Total and free MPA, MPAG, and AcMPAG concentrations in plasma and urine were measured by use of HPLC with tandem mass spectrometry detection. RESULTS: Total MPA area under the plasma concentration-time curve (AUC) from 0 to 12 hours decreased significantly after rifampin coadministration (17.5% decrease [95% confidence interval (CI), 5.18%-29.9%]; P=.0234). This was mainly a result of a decrease in total MPA AUC from 6 to 12 hours (32.9% decrease [95% CI, 15.4%-50.4%]; P=.0078), representing decreased enterohepatic recirculation. Free MPA AUC from 6 to 12 hours decreased significantly, by 22.4% (95% CI, 4.71%-49.5%; P=.0391). Total MPAG and AcMPAG AUC from 0 to 12 hours increased by 34.4% (95% CI, 13.5%-55.4%; P=.0156) and 193% (95% CI, 30.3%-355%; P=.0078) respectively. Urinary recovery of MPAG and AcMPAG increased significantly (P=.0078), but renal clearance of these glucuronides did not change after rifampin coadministration. CONCLUSION: This study demonstrates an interaction between mycophenolate mofetil and rifampin, which is a result of induction of MPA glucuronidation and possibly also rifampin-associated alterations in MRP2-mediated transport of MPAG and AcMPAG. This interaction should be taken into account when rifampin or other drugs influencing pregnane X receptor activity are coadministered with mycophenolate mofetil.     

Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid

BACKGROUND: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. METHODS: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. RESULTS: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA. CONCLUSIONS: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.     

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.     

The impact of UGT1A8, UGT1A9, and UGT2B7 genetic polymorphisms on the pharmacokinetic profile of mycophenolic acid after a single oral dose in healthy volunteers

We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 -275/-2152 selected among 305 individuals genetically screened for UDP-glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype -1248A>G, -1241T>C, -1054T>C, -842G>A, -268A>G, -102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high-performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 -275/-2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration-time curve (AUC6-12 h/AUC0-12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 -275/-2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA C(max) values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.     

Quantitative measurement of porphobilinogen in urine by stable-isotope dilution liquid chromatography-tandem mass spectrometry

BACKGROUND: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity. METHODS: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[(13)C]pyrolle-3-propanoic acid ([2,4-(13)C]PBG; 2.75 microg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-(13)C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-(13)C]PBG was 1.0 min in a 2.3-min analysis. RESULTS: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2-3.5% and 2.6-3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (S(y/x) = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02-0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG < or =0.5 by LC-MS/MS, 8 (9.4%) had values > or =2.0 mg/24 h by the anion-exchange method (mean +/- SD, 4.3 +/- 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method. CONCLUSIONS: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.     

Method for the determination of total homocysteine in plasma and urine by stable isotope dilution and electrospray tandem mass spectrometry.

BACKGROUND: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. METHODS: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 microL of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d(4) was 1.5 min in a 2.5-min analysis. RESULTS: Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S(y|x) =1.595 micromol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S(y|x) =1.146 micromol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/L). CONCLUSIONS: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.     

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.     

Usefulness of mycophenolic acid monitoring with PETINIA for prediction of adverse events in kidney transplant recipients

Background Therapeutic drug monitoring of mycophenolic acid (MPA) is required to optimize the immunosuppressive effect and minimize toxicity. We validated a new particle-enhanced turbidimetric inhibition immunoassay (PETINIA) for the determination of MPA levels and evaluated the relationship of MPA trough level with drug-related adverse events. Methods PETENIA and liquid chromatography-mass spectrometry (LC-MS) were used to determine MPA concentrations from 54 kidney transplant recipients (KTRs). Agreement between PETINIA and LC-MS results was assessed by Passing-Bablok regression and the Bland-Altman plot method. The association of adverse events with MPA trough level obtained by PETINIA was analyzed. Results PETINIA revealed a good agreement with the LC-MS; Regression analysis gave an equation of y?=?1.27x???0.12 (r²?=?0.975, p?p?=?0.007). MPA trough level predicted adverse events with a sensitivity of 77.8% and a specificity of 86.7% using a cut-off level of 5.25?mg/L. Conclusions Good correlation between the two methods indicates that PETINIA is an acceptable method for the monitoring of MPA therapeutic levels. Furthermore, MPA trough level obtained by PETINIA is a useful monitoring tool to minimize toxicity in KTRs.     

Mycophenolate mofetil in stem cell transplant patients in relation to plasma level of active metabolite

OBJECTIVES: To investigate mycophenolate mofetil (MMF) plasma levels and impact on acute graft versus host disease (aGvHD) after stem cell transplantation (SCT).METHODS: SCT patients (n = 14) with aGvHD (>/= II) receiving MMF (1-3 g/d) in addition to cyclosporine, prednisolone, and methotrexate for aGvHD prophylaxis were investigated. Plasma levels of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) were determined by high-performance liquid chromatography.RESULTS: Overall median steady state pre-dose plasma MPA concentration was 0.47 mg/L and increased within 75 min after administration to 1.64 mg/L. In comparison to patients with skin aGvHD, patients with gut aGvHD had lower MPA concentrations, both pre-dose (p = 0.16) and after 75 min, (p = 0.02). All 7 patients with skin aGvHD but only 2 patients with gut aGvHD responded to MMF. Overall, the pre-dose plasma MPA concentration was significantly (p = 0.007) greater in responders (n = 9) than in non-responders (n = 5).CONCLUSION: MMF seems to be an effective treatment for aGvHD in SCT patients particular in those patients without gut involvement.     

High-performance liquid chromatographic method for mycophenolic acid and its glucuronide in serum and urine

OBJECTIVE: To develop a simple analytical method for monitoring serum and urine concentrations of mycophenolic acid (MPA), an active metabolic constituent of the immunosuppressive pro-drug mycophenolate mofetil, and its glucuronide. METHODS: Serum samples were prepared by solid-phase extraction (SPE), while urine samples were simply diluted with water. Serum was added to an SPE cartridge, then washed twice with 5% methanol solution. The analytes were eluted with methanol containing benzoic acid as internal standard for mycophenolic acid glucuronide (MPAG). The resultant eluate was directly injected into a high-performance liquid chromatograph (HPLC) to determine MPAG. For the assay of MPA, the remaining eluate was dried under nitrogen and resolved in a mixture of acetonitrile and 20 mM phosphate buffer (pH 3.0). RESULTS: The present methods were reproducible and accurate based on the intra- and inter-assay, and had detection limits of 0.225 microg/mL for MPA and 9.0 microg/mL for MPAG. The present methods enabled us to monitor the time course of changes in the concentrations of MPA and MPAG in serum and urine in a patient with a renal transplant during 12 h after ingestion of mycophenolate mofetil. CONCLUSION: The HPLC method described should be useful for the routine monitoring of serum and urine concentrations of MPA and MPAG during immunosuppressive medication for renal transplantation.     

Validation of a high-performance liquid chromatography method for the measurement of mycophenolic acid and its glucuronide metabolites in plasma

OBJECTIVES: The need for therapeutic drug monitoring of the immunosuppressant mycophenolic acid is becoming more evident. This paper describes a simple high-performance liquid chromatography procedure for the simultaneous quantitation of mycophenolic acid (MPA) and its glucuronide metabolites in plasma using protein precipitation followed by HPLC analysis with isocratic elution and UV detection. DESIGN AND METHODS: The performance of this method is compared to the EMIT 2000 MPA immunoassay (Dade Behring Diagnostics Inc., Cupertino, California, USA). RESULTS AND CONCLUSION: Intra-assay precision and accuracy of calibrators were determined for MPA at 0.5 and 20 mg/L, MPAGe at 5 and 200 mg/L, and MPAGa at 2.5 and 100 mg/L and showed coefficients of variation of less than 5.0% and biases of less than 14.0%. Inter-assay precision and accuracy of quality control samples were determined for MPA at 2 and 15 mg/L, MPAGe at 20 and 150 mg/L and showed CVs of less than 5.0% and biases of less than 14%. The lower limit of quantitation of the method was determined for MPA at 0.25 mg/L, MPAGe at 0.5 mg/L, and MPAGa at 0.25 mg/L and showed CVs of less than 19% and biases of less than 20%. This method, compared to the EMIT 2000 MPA immunoassay, showed a linear regression analysis relationship of EMIT = 0.973 HPLC + 0.55 (r(2) = 0.851), and was determined to be suitable for therapeutic drug monitoring and pharmacokinetic studies of MPA.     

Cyclosporine alters correlation between free and total mycophenolic acid in kidney transplant recipients in the initial phase

What is known and Objective: The factors affecting the pharmacokinetics of free mycophenolic acid (MPA) and its phenolic glucuronide (MPAG) are still unclear. The aim of this study was to evaluate the influence of cyclosporine on the pharmacokinetics of free MPA and MPAG. Methods: Seventy‐seven kidney transplant recipients (23 were in an initial phase and 54 in a stable phase; 41 were treated with cyclosporine and 36 with tacrolimus) were enrolled. Free and total MPA and MPAG were determined using HPLC. The correlations between free and total predose concentrations (C₀) of MPA or MPAG were evaluated separately in patients receiving calcineurin inhibitor medications. Results and Discussion: Serum concentration of albumin was lower in the initial phase than in the stable phase. A higher ratio of free MPAG C₀ to free MPA C₀ was observed in cyclosporine‐treated than tacrolimus‐treated kidney transplant recipients. Free MPA C₀ correlated weakly with total MPA C₀ in kidney transplant recipients treated with cyclosporine in the initial phase (ρ = 0·53, P = 0·06). What is new and Conclusion: Cyclosporine increased the ratio of free MPAG C₀ to free MPA C₀ and varied the free fraction of MPA in the hypoalbuminaemic kidney transplant recipients in the initial phase.