Navigation Links
A new LC/MS/MS Research Method for Rapid Quantitation of Five Immunosuppressant Drugs, including Mycophenolic Acid (MPA)

Purpose

A previously published application note in this series describes an LC/MS/MS method for the simultaneous determination of four immunosuppressant drugs: Cyclosporin A (CsA), Tacrolimus (TAC, aka FK-506), Sirolimus (SIR, aka Rapamycin) and Everolimus (EVE, aka RAD-001) (1, 2). The method is based on a simple and robust hardware configuration and shows to be fast and simple, without compromising precision and accuracy. Since this method was first described, there has been considerable interest in extending it to include a fifth immunosuppressant drug, Mycophenolic Acid (MPA, see structure in Figure 1).

MPA is the pharmacologically active form of the immunosuppressant prodrug Mycophenolate Mofetil. Because its pharmacological action differs significantly from CsA, TAC, SIR, and EVE, MPA is frequently co-prescribed as part of an immunosuppressant drug cocktail. Due to the chemical structure of MPA and the strong presence of some of its metabolites -most notably the Glucuronide metabolite, MPAGseveral authors have voiced their concern about the risk of overestimating the MPA concentration. It has been shown that chromatographic separation is essential to accurately measure MPA concentration in plasma by eliminating the interfering effect of MPAG (3, 4).

Overview

In this research note, minor modifications are described that extend the original method proposed in reference (1) to include MPA and determine the entire panel of 5 immunosuppressants on the Applied Biosystems API 3200TM LC/MS/MS Mass Spectrometer. With these modifications, the same hardware configuration can be used to determine CsA, TAC, SIR, and EVE in blood and MPA in plasma, without compromising sensitivity and specificity. Quantitation of MPA metabolites (mainly MPAG) can be included if desired.

Depending on the instrumental configuration and the up-stream sample preparation step, the required time for the entire analytical procedure significantly influences the final overall sample throughput (5).

As with the original method, a fast and inexpensive on-line solid-phase extraction (SPE) is performed through a twodimensional liquid chromatography configuration. Using this configuration, sample clean up effectively minimizes any ion suppression interference during the MRM measurements. This permits the use of Cyclosporin D (CsD) as an internal standard for quantitation of all 5 immunosuppressant drugs included in the extended panel.

Experimental conditions

Samples were prepared for MPA measurements by adding 100 μL of a diluting solution* to either 50 μL of either plasma samples, calibrators or controls, This mixture was then vortex mixed for 30 seconds. After centrifugation at 13000 rpm x 5 min., 100 μL of supernatant were transferred to either an autosampler vial or a microtiter plate.

The hardware configuration included an Applied Biosystems/MDS SCIEX API 3200TM Triple Quadrupole Mass Spectrometer equipped with a Turbo VTM source. This source operates in positive ion mode at a voltage of +5500 volts and with a Turbo V gas heated at 650 C.

Multiple Reaction Monitoring (MRM) measurements were made using declustering potential (DP) and collision energy (CE) values, which were automatically optimized by the Analyst software for each of the analytes. DP values ranged between 40 and 140 V; CE values were between 30 and 65 eV.

Two-dimension chromatography was performed through a split arrangement of the modules of the LC pumping system (either a Perkin Elmer Series 200 Micro Pump, an Agilent 1100 Binary Pump with the pump heads disconnected from the mixing tee or a Shimadzu Prominence or 10Dvp pumps) and a computer-controlled Valco Valve (10-port, 2 positions) plumbed as illustrated in Figure 2. www.appliedbiosystems.com First dimension chromatography was accomplished via an Applied Biosystems POROS R1/20, 2.1 x 30 mm perfusion-column. Second dimension chromatography was performed by a Phenomenex Luna 5 μm Phenyl- Hexyl, 2 x 50 mm column (p.n. 00B- 4257-B0) housed in an oven at 60 C.

Diluting solution consisted of 80% methanol with 1% ZnSO4 and 200 ng/mL of Cyclosporin D as internal standard. 20 ng/mL Ascomycin is added when TAC, SIR and EVE are quantified in the same sample

Results and discussion

In relation to the other immunosuppressant drugs in the 5-drug panel, there are several analytical features unique to MPA. These are discussed below.

Despite the presence of a free acidic moiety, which should make MPA very sensitive when ionized in negative ion mode, the sensitivity in positive ion mode is higher. Several papers have demonstrated that the molecule is quite labile in the ionization process (5).

Fragmentation of the MPA precursor ion (m/z 321) results in an MS/MS spectrum as shown in Figure 3, with the most intense fragment ion at m/z 207.1.

MPA is predominantly metabolized to the glucuronide form (MPAG). This glucuronidated form is at a much higher concentration (around one order of magnitude greater) than the residual MPA in the specimen.

Despite the mild source-declustering conditions, the MPAG metabolite is prone to fragmentation in the sourceinterface region. As shown in Figure 4, the MS spectrum for the MPAG metabolite shows two prominent ions corresponding to the ammoniumadduct ion [M+NH4]+ (m/z 514.2) and the glucuronide-depleted ion, which is the original MPA protonated ion [M+H]+ (m/z 321.2) produced by in-source fragmentation.

The MS/MS spectrum of MPAG (Figure 5) is directly comparable with that of pure MPA (Figure 5 v Figure 3), since the glucuronide moiety becomes a neutral loss when MS/MS is performed.(Both are dominated by the fragment ion at m/z 207.2.)

In setting the quantitation measurement through the resulting MRM transitions (321.3 > 207.2 for MPA and 514.3 > 207.2 for MPAG), it is important to distinguish that the chromatographic peak seen at the transition 514.3 > 207.2 and corresponding to MPAG is coincident with a chromatographic peak at the same retention time, but obtained through the transition (321.3 > 207.2) specific for MPA (Figure 6). This is determined to be MPAG in-source fragmenting into free MPA, making it clear that chromatographic separation is extremely important in segregating the endogenous MPA in the specimen from the MPA derived from the in-source fragmentation of MPAG.

The modified configuration described here makes it possible to include the analytes CsA, TAC, SIR, EVE, and the additional internal standard (CsD) into the method and quantitate them simultaneously whenever they are present in the same specimen. (MPA is generally analyzed in plasma while the rest of the compounds are quantitated in whole blood). Figure 7 shows the applicability of the method to real samples. In these optimized conditions, the total run time is approximately 5 minutes per sample.

Figure 8 describes the obtained linearity for the samples in the range 0.5 15 ug/mL and Figure 9 gives proof of the intra-run reproducibility on a quality control plasma sample (Level I, Recipe-Munich, Reference Value 0.48 ug/mL). Deviation from the mean value is restricted to 0.5 %.The estimated detection limit on the API 3200 using this protocol is approximately 0.03 μg/mL plasma.

Figure 10 shows the correlation obtained when MPA quantitation is compared with a commercially available HPLC-UV assay.

Conclusion

The modified method described in this application note (1, 2) has been demonstrated to be:

Rapid. It is suitable for quantifying either MPA, and/or the other immunosuppressant drugs (CsA, TAC, SIR, EVE).

Flexible. With the same hardware setting and configuration, the method can handle whole blood, for CsA, TAC, SIR, and EVE and plasma for MPA and MPAG.

Accurate. MPAG, usually present at significant concentrations, does not interfere with the MPA quantitation results.

Fully under control of the Analyst acquisition software. All the peripheral modules are under control of the fullinclusive Acquisition Method.

Effective in reducing the solvent consumption. The programmed operation of the LC peripheral units enables the high flow-rate regime in the firstdimension LC just for the time needed for the sample clean up.

References

1. Applied Biosystems Application, Publication: MS-AP234.

2. T. Koal, M. Deters, B. Casetta, and V. Kaever, J. Chromatogr B, 805 (2004) 215-222.

3. B. Atcheson, P.J. Taylor, D.W. Mudge, D.W. Johnson, I. Pillans, and S.E.Tett. J. Chromatogr, B. Analyt Technol Biomed Life Sci, Jan 2004; 799(1): 157-63.

4. F. Streit, M. Shipkova, V.W. Armstrong, and M. Oellerich, Clinical Chemistry 50:1 152159 (2004).

5. T.M. Annesley, L.T. Clayton, Clinical Chemistry 51:5 872877 (2005).

Acknowledgements

Applied Biosystems/MDS SCIEX acknowledges our collaborators, Jan Lembke and Uta Ceglarek of the Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital, Leipzig (Germany), Bruno Casetta of Applied Biosystems Monza (Milano- Italy), and Daniel Blake of Applied Biosystems, Warrington (UK) for providing the method, samples, and data for this application note.


'"/>

Source:


Page: All 1 2 3 4 5

Related biology technology :

1. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
2. The Analysis of Polycyclic Aromatic Hydrocarbons (PAHs) by LC/MS/MS Using an Atmospheric Pressure Photoionization Source
3. HPLC/MS/MS Analysis of Bitter Acids in Hops and Beer
4. Simultaneous Determination of Residues of Approximately 100 Pesticides and Metabolites in Fruit and Vegetables by LC/MS/MS
5. The combination of monolithic columns and modern HPLC/MS/MS instrumentation for high chromatographic throughput and sensitive detection of beta-agonists
6. Determination of Polar Organophosphorus Pesticides in Aqueous Samples by Direct Injection Using HPLC/MS/MS
7. Detection of nitrofurane metabolites in food with HPLC/MS/MS
8. Simultaneously characterizing and quantifying chloramphenicol and its metabolite using LC/MS/MS
9. Determination of the Chloramphenicol residues in milk and milk products using LC/MS/MS
10. High sensitivity quantitation of metabolites of nitrofuran antibiotics in animal tissue using LC/MS/MS
11. A Robust LC/MS/MS Method for the Quantitation of Very Long Chain Fatty Acids for Research into the Characterization of Peroxisomal Disorders
Post Your Comments:
(Date:10/19/2014)... 19, 2014 The Asia-Pacific Bromine ... in Asia-Pacific with analysis and forecast of revenue. ... Pacific Bromine Market report, to get an idea ... a glimpse of the segmentation in the Asia-Pacific ... and figures. , http://www.micromarketmonitor.com/market/asia-pacific-bromine-6741503144.html , ...
(Date:10/19/2014)... October 19, 2014 The report ... & Homeland Security), Technology, Payload, Type & by ... segments the global unmanned ground vehicle market into ... forecasting of market sizes. The report also identifies ... opportunities impacting it along with the adoption trends. ...
(Date:10/18/2014)... The Asian arthroscopic devices report defines and ... forecast of revenue. The arthroscopic devices market in Asia ... at a CAGR of 6.5% from 2013 to 2018. ... devices market, to get an idea of the in-depth ... segmentation of arthroscopic devices market in the same region, ...
(Date:10/18/2014)... (PRWEB) October 18, 2014 According to ... Resource & Well Type - Global Trends & Forecasts ... estimated $31 billion in 2011. This value is expected ... billion by 2017, with 10% CAGR during the same ... hydraulic fracturing production market in terms of hydraulic horse ...
Breaking Biology Technology:The Asia-Pacific Bromine Market is estimated to grow to $4,080.1 million by 2018 - New Report by MicroMarket Monitor 2The Asia-Pacific Bromine Market is estimated to grow to $4,080.1 million by 2018 - New Report by MicroMarket Monitor 3Unmanned Ground Vehicle (UGV) Market Estimated to Reach $8.26 Billion by 2020 - New Research Report by MarketsandMarkets 2Unmanned Ground Vehicle (UGV) Market Estimated to Reach $8.26 Billion by 2020 - New Research Report by MarketsandMarkets 3Unmanned Ground Vehicle (UGV) Market Estimated to Reach $8.26 Billion by 2020 - New Research Report by MarketsandMarkets 4Unmanned Ground Vehicle (UGV) Market Estimated to Reach $8.26 Billion by 2020 - New Research Report by MarketsandMarkets 5The Asian arthroscopic devices market is estimated to grow to $712.3 million by 2018 - New Report by MicroMarket Monitor 2The Asian arthroscopic devices market is estimated to grow to $712.3 million by 2018 - New Report by MicroMarket Monitor 3The Asian arthroscopic devices market is estimated to grow to $712.3 million by 2018 - New Report by MicroMarket Monitor 4Hydraulic Fracturing Market Projected To Reach $64 Billion by 2017 - New Report by MarketsandMarkets 2Hydraulic Fracturing Market Projected To Reach $64 Billion by 2017 - New Report by MarketsandMarkets 3Hydraulic Fracturing Market Projected To Reach $64 Billion by 2017 - New Report by MarketsandMarkets 4
... Corporation,(Nasdaq: NEOG ) announced today that its revenues ... 31, increased 26% from the previous year,s first,quarter to ... year,s $3,011,000 to $3,733,000, or to $0.25 per share ... Neogen,s first quarter revenues, net income, and earnings ...
... for antibody therapeutics, BASEL, Switzerland, Sept. 24 ... has completed the acquisition of 100% of the ... (TSX: ARI). On,September 19th, the Ontario Superior Court, ... the security holders and announced in a,press release ...
... Journal to Honor http://www.animetrics.com/ [Animetrics] at "Top 100 Private ... ... September 24, 2008 -- Animetrics® Inc., a leading developer of 3D ... named one New Hampshire,s 10 private companies to watch by Business ...
Cached Biology Technology:Neogen Reports 26% Increase in Revenues 2Neogen Reports 26% Increase in Revenues 3Neogen Reports 26% Increase in Revenues 4Neogen Reports 26% Increase in Revenues 5Neogen Reports 26% Increase in Revenues 6Roche completes acquisition of ARIUS 2Animetrics Inc. Named One of Business NH Magazine's '10 Companies to Watch' for its Significant Revenue Growth 2
(Date:10/15/2014)... , Oct. 15, 2014 Sandata ... for home and community-based care, today announced it ... implementing Sandata,s Santrax® Electronic Visit Verification™ Solution (EVV™) ... is a home health company founded in 1996 ... . The study details ...
(Date:10/15/2014)... rapidly and to an unexpected extent. The outbreak does ... the virus shows a new disease dynamic in regions, ... reason, the German National Academy of Sciences Leopoldina, acatech ... the Union of the German Academies of Sciences and ... today. , In the statement the academies call for ...
(Date:10/15/2014)... VILNIUS, Lithuania , Oct. 15, 2014 /PRNewswire/ ... identification technologies, today announced the availability of the ... . VeriLook Surveillance 3.0 provides real-time biometric face ... multiple high-resolution digital surveillance cameras. The new version ... detects and differentiates people from objects while they ...
Breaking Biology News(10 mins):Sandata Announces Case Study with Quality Care Services, Inc. 2Academies call for consequences from the Ebola virus epidemic 2Academies call for consequences from the Ebola virus epidemic 3Academies call for consequences from the Ebola virus epidemic 4Academies call for consequences from the Ebola virus epidemic 5VeriLook Surveillance 3.0 SDK Identifies Faces and Moving Objects, Differentiates Pedestrians from Other Moving Objects in Video Surveillance Systems 2VeriLook Surveillance 3.0 SDK Identifies Faces and Moving Objects, Differentiates Pedestrians from Other Moving Objects in Video Surveillance Systems 3
... 2011 /PRNewswire-Asia/ -- Clearbridge Accelerator announced today that it ... venture capitalist based in the Silicon Valley. Mr Draper, ... Draper Fisher Jurvetson (DFJ), is well known for making ... Clearbridge Accelerator is a technology incubator that ...
... researchers have developed the first lab-on-a-chip sensor to provide fast ... The sensor is both environmentally and child friendly, and ... a UC researcher is leading a long-term health study on ... University of Cincinnati researchers to create a sensor that provides ...
... of the newest edition of its authoritative reference for ... edition, is available in both print and, for the ... of the Manual of Clinical Microbiology marks a significant ... Editor-in-Chief James Versalovic of Texas Children,s Hospital. "For the ...
Cached Biology News:Singapore Technology Incubator Receives Investment from Silicon Valley "Super-Angel" 2Singapore Technology Incubator Receives Investment from Silicon Valley "Super-Angel" 3Singapore Technology Incubator Receives Investment from Silicon Valley "Super-Angel" 4Singapore Technology Incubator Receives Investment from Silicon Valley "Super-Angel" 5Singapore Technology Incubator Receives Investment from Silicon Valley "Super-Angel" 6New UC sensor promises rapid detection of dangerous heavy metal levels in humans 2New UC sensor promises rapid detection of dangerous heavy metal levels in humans 3New edition of Manual of Clinical Microbiology offers digital access 2
Anti-Goat HRP-DAB Cell & Tissue Staining Kit...
... The Light Diagnostics Cytomegalovirus (CMV) pp65 Antigenemia ... qualitative identification of lower matrix protein pp65 ... Kit Components: ... One bottle containing 220 mL of buffered ...
... The CultureWell cell culture ... for preparing cultured cells for ... uses medical-grade silicone gaskets preassembled ... cell culture plates. The entire ...
nuclear cap binding protein subunit 1, 80kDa, mRNA...
Biology Products: