1. GUY’S & St THOMAS’ FOUNDATION TRUST A simple and rapid method for quantifying total and free Mycophenolic acid and its glucuronide metabolite using Electrospray LC-MS/MS A. Sankaralingam 1, R Swaminathan 1, DP Cooper 2 1)Chemical Pathology,Guy’s & St Thomas’ NHS Foundation Trust, London,UK 2)Clinical Application Group, Waters UK Ltd, Manchester, UK   MPA is increasingly used in all new transplants to minimize the use of  Scientific evidence support the therapeutic monitoring of MPA for effective graft survival and prevention of acute rejection  MPA is extensively bound to albumin in patients with normal re renal and liver function  MPA has significant variation   AIM  To develop a rapid and simple method for total and free MPA  To evaluate and introduce the method to routine service Contact details: arun.sankaralingam@gstt.nhs.uk   To evaluate and introduce the method to routine service 0 2 4 6 8 10 12 024681012 -0.4 -0.2 0 0.2 0.4 0.6 1 1.2 051015 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 051015 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 01015 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 051015 0 2 4 6 8 10 12 024681012 0 2 4 6 8 10 12 024681012 -sparing maintenance regimens in kidney transplant that helps to minimize the patient 1. There is 2. The current approach is measurement of total MPA concentration. However some advocate the m 3. for routine quantification of both total and free MPA. The on for total-MPA is similar to the method 80uL of 0.1M zinc sulphate solution and extracted in 200uL was performed with 10uL of the supernatant using a 2.1 x 50mm symmetry C8 3.5µm analytical column, held at ambient temperature, and gradient elution at a flow rate of 0.6ml/min. The mobile p water containing 2mM ammonium acetate and 0.1% formic -MPA, 4 Q1/Q3 transition in positive ionisation mode was monitored at m/z 338.5/207.0, 514.5/207.7 and 438.5/207.0 for MPA, MPAG and MPAC respectively. The injection to injection time was 3.5min. The assay for total-MPA was calibrated -house calibrators -MPA and MPAG respectively. The linear calibration range for total-MPA, free-MPA and MPAG were 0--500ug/L (r=0.9995) and 0-500mg/L (r=0.996) respectively. Total-MPA: The Intra-day and Inter- 4.65– 10.4% and 4.7– 7.7% respectively. The mean analytical recovery was 101.6%. The lower limit of Proficiency Testing Scheme- International Ltd, analysed using this method showed an method group which included HPLC/UV, LC-MS and LC- MS/MS. The method is simple and reliable and has been adapted for routine provision of service. Fig 1 MRM chromatograms of a mixture containing MPAG (rt 1.33), acyl-MPAG(rt 1.66), MPA & MPAC(rt 1.73) Results Fig 2 calibration curve for tMPA ranging from 0-100mg/L Fig 3 calibration curve for MPAG spanning from 0 – 500mg/L Fig 4 calibration curve for fMPA ranging from 0 – 500ug/L Fig 5 linearity of tMPA assay. Drug free plasma spiked with MPA was serially diluted and concentration measured using commercial calibrant Fig 6 Bland-Altman difference plot EQA vs Guy’s LC-MS/MS Introduction  MPA is increasingly used in all new transplants to minimize the use of calcineurin inhibitors and steroids  Scientific evidence support the therapeutic monitoring (TDM) of MPA for effective graft survival and prevention of acute rejection  MPA is extensively bound to albumin in patients with normal renal and liver function  The extent of metabolism of MPA varies among individuals. Hence MPAG, glucuronide metabolite, measurement is useful for optimising the therapy 5  MPAG is prone to fragmentation in the source to form MPA. The potential for interference from this effect can be eliminated by chromatographically separating MPAG from MPA (Fig1)  For the accurate measurement of MPAG, other active minor metabolite, acyl-MPA- glucuronide should be separated from MPAG (Fig 1)  Current approach is the measurement of total MPA(tMPA) concentration for TDM  However some advocate the measurement of pharmacologically active free MPA (fMPA) Abstract Mycophenolate mofetil (MMF) has become an integral component of toxicity-sparing maintenance regimens in kidney transplantation that helps to minimize the patient exposure to calcineurin inhibitors and steroids 1.There is scientific evidence supporting the therapeutic monitoring of mycophenolic acid(MPA), the active moiety of mycophenolate mofetil(MMF), for the effective graft survival and prevention of acute rejection 2. The current approach is measurement of total MPA concentration. However some advocate the measurement of clinically useful free mycophenolic acid, the pharmacologically active form, in hypoalbuminemic patients 3. Here we have developed a rapid and simple method suitable for routine quantification of both total and free MPA. The sample preparation for total-MPA is similar to the method that is currently used for cyclosporin, tacrolimus & sirolimus in our laboratory. Plasma (20μL) was treated with 80μL of 0.1M zinc sulphate solution and extracted in 200μL of methanol containing 10mg/L MPAC (MPA carboxy butoxy ether) as an internal standard. The chromatography was performed with 10uL of the supernatant using a 2.1 x 50mm symmetry C8 3.5μm analytical column, held at ambient temperature, gradient elution at a flow rate of 0.6ml/min. The mobile phase consisted of methanol and water containing 2mM ammonium acetate and 0.1% formic acid. Separation of MPA and the glucuronide metabolite (MPAG) was achieved in less than 2mins. For free-MPA, plasma was subjected to ultrafiltration using a Centrifree(Millipore) device 4. 50μL of the ultrafiltrate was mixed with 100μL methanol containing 2ug/L MPAC. The Q1/Q3 transition in positive ionisation mode was monitored at m/z 338.5/207.0, 514.5/207.7 and 438.5/207.0 for MPA, MPAG and MPAC respectively. The injection to injection time was 3.5min. The assay for total-MPA was calibrated using a commercial one point calibrant. In- house calibrators were used for free-MPA (5 points) and MPAG (6 points). The linear calibration range for total-MPA, free-MPA and MPAG were 0-100mg/L (r=0.9999), 0-500ug/L (r=0.9995) and 0-500mg/L (r=0.996) respectively. Total-MPA: The Intra-day and Inter-day imprecision were 4.65-10.4% and 4.7-7.7% respectively. The mean analytical recovery was 101.6%. The lower limit of quantitation was 0.3mg/L. The external quality samples (n=15), from International Proficiency Testing Scheme-Analytical Services International Ltd, analysed using this method showed an excellent correlation (slope=0.9315; r=0.997) with the method group which included HPLC/UV, LC-MS and LC-MS/MS. The method is simple and reliable and has been adapted for routine provision of service. Aim  To develop a rapid and simple method for total and free MPA and the glucuronide metabolite, MPAG  To evaluate and introduce the method to routine service Method Total MPA (tMPA) Plasma (20μL) & 0.1m zinc sulphate (80μL) Vortex mix for 5 secs Extract with 200μL of methanol containing 10mg/L MPAC (carboxy butoxy ether of MPA), the internal standard Vortex mix and centrifuge for 5mins at 15800 x g Chromatography is carried out with 10μL of the supernatant Free MPA (fMPA) Ultrafilter the plasma Mix 50μL of the ultrafiltrate with 100μL of methanol containing 2μg/L MPAC (internal standard) Vortex mix and centrifuge Chromatography is carried out with 20μL of the supernatant Multi point in-house calibrators were used for tMPA (6 points), fMPA (5 points) and MPAG (6 points) to construct the calibration curve(Fig 2,3&4) Single point commercial calibrant (chromsystems) was used for the quantitative analysis of tMPA in the comparison study (Fig 6) Chromatographic separation was achieved with 2.1 x 50mm Symmetry C8 column held at ambient temperature Gradient elution was with methanol and water containing 2mM ammonium acetate and 0.1% formic acid at flow rate 0.6ml/min Q1/Q3 transition in positive ionisation mode was monitored at m/z 338.5/207.0 (MPA), 514.5/207.7 (MPAG) and 438/207.0 (MPAC) using a WATERS Quattro MPAG Acyl-MPAG MPAC MPA Performance Characteristics for total MPA Calibration range (mg/L) 0 – 100 Mean recovery(%) 101.6 Functional sensitivity(mg/L) 0.3 Inter-assay CV(%) 4.6 – 10.4 Intra-assay CV(%) 4.7 – 7.7 338.5>207.0 438.5>207.0 514.5>207.7 Summary  Very simple method that can be adapted for routine service  LC-MS/MS has both high throughput and sensitivity/specificity unlike HPLC (low throughput) and immunoassay (interference from metabolites)  MPAG, glucuronide metabolite of MPA, can be measured in the same run Conclusion Therapeutic measurement of Mycophenolic acid is made simple with LC-MS/MS References 1) A prospective randomised trial of tacrolimus in combination with sirolimus or mycophenolate mofetil in kidney transplantations: results at 1 year Robert Mendez et al Transplantation 2005 Aug 15; 80(3): 303-309 2) Determination of mycophenolic acid levels after renal transplantation Borrows R et al Therapeutic Drug Monitoring 2005 Aug; 27(4): 442-450 3) Free mycophenolic acid should be monitored in renal transplant reciepients with hypoalbunemia Atcheson BA et al Therapeutic Drug Monitoring 2004 Jun; 26(3): 284-286 4) Validation of a Rapid and Sensitive liquid chromatography-Tandem mass spectrometry method for free and total mycophenolic acid Streit F et al Clinical Chemistry 2004; 50: 152-159 5) Clinical pharmacokinetics of mycophenolate mofetil Bullingham RE et al Clin Pharmacokinet 1998; 34: 429-455