Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and determination of the matrix effect

A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5–50.0 ng/ml (r2 > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30–40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration. A simple, specific, and sufficiently sensitive liquid chromatography-tandem mass spectrometry (negative-ion electrospray ionization) methodology to determine mevalonic acid (MVA) in human plasma is described, and its application to the analysis of rat plasma MVA levels after rosuvastatin administration is demonstrated. The method was validated over the linearity range of 0.5–50.0 ng/ml (r2 > 0.99) using deuterated MVA as an internal standard. The lower limit of quantification was 0.5 ng/ml. The assay procedure involved the isolation of MVA from plasma samples using solid-phase extraction. Chromatographic separation was achieved on a HyPurity Advance column with a mobile phase consisting of ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, v/v). Excellent precision and accuracy were observed. MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing conditions. The recovery observed was low, which was attributable to a significant matrix effect. A significant decrease (30–40%; P < 0.05) was observed in rat plasma MVA levels after rosuvastatin administration. In the biosynthesis of cholesterol, the conversion of HMG-CoA to mevalonic acid (MVA) by HMG-CoA reductase is an early and rate-limiting step (1Parker T.S. McNamara D.J. Brown C.D. Kolb R. Ahrens E.H. Tobert J. DeSchepper P.J. Plasma mevalonate as a measure of cholesterol synthesis in man..J. Clin. Invest. 1984; 74: 795-804Crossref PubMed Scopus (134) Google Scholar, 2Popljak G. Boehm C. Parker T.S. Edmond J. Edwards P.A. Fogelman D.A. Determination of' mevalonate in blood plasma in man and rat. Mevalonate tolerance tests in man..J. Lipid Res. 1979; 20: 716-728Abstract Full Text PDF PubMed Google Scholar, 3Yoshida T. Honda A. Tanaka N. Matsuzaki Y. He B. Osuga T. Kobayashi N. Ozawa K. Miyazaki H. Simultaneous determination of mevalonate and 7 alpha-hydroxycholesterol in human plasma by gas chromatography-mass spectrometry as indices of cholesterol and bile acid biosynthesis..J. Chromatogr. 1993; 613: 185-193Crossref PubMed Scopus (59) Google Scholar). The statin class of drugs, such as simvastatin, atorvastatin, and rosuvastatin, act on HMG-CoA reductase, resulting in the inhibition of MVA biosynthesis (Fig. 1) (4Scoppola A. Maher V.M. Thompson G.R. Rendell N.B. Taylor G.W. Quantitation of plasma mevalonic acid using gas chromatography-electron capture mass spectrometry..J. Lipid Res. 1991; 32: 1057-1060Abstract Full Text PDF PubMed Google Scholar, 5Naoumova R.P. Dunn S. Rallidis L. Muhana O.A. Neuwirth C. Rendell N.B. Taylor G.W. Thompson G.R. Prolonged inhibition of cholesterol synthesis explains the efficacy of atorvastatin..J. Lipid Res. 1997; 38: 1496-1500Abstract Full Text PDF PubMed Google Scholar). Understanding the reason for increased cholesterol levels and interindividual variability in response to statin therapy can lead to better and monitored pharmacotherapy (6O'Neill F.H. Patel D.D. Knight B.L. Neuwirth C.K. Bourbon M. Soutar A.K. Taylor G.W. Thompson G.R. Naoumova R.P. Determinants of variable response to statin treatment in patients with refractory familial hypercholesterolemia..Arterioscler. Thromb. Vasc. Biol. 2001; 21: 832-837Crossref PubMed Scopus (62) Google Scholar). Because the reduction of MVA levels is an indirect measure of decreased cholesterol levels, MVA can be used as a biomarker to measure the extent of statin activity. A large variety of methods have been published for MVA estimation in urine and plasma. These involve enzyme immunoassay (7Makoto H. Akio H. Hideki H. Hirotsugu U. Takashi K. Enzyme immunoassay of urinary mevalonic acid and its clinical application..Clin. Chem. 1998; 44: 2152-2157Crossref PubMed Scopus (10) Google Scholar), radioimmunoassay (2Popljak G. Boehm C. Parker T.S. Edmond J. Edwards P.A. Fogelman D.A. Determination of' mevalonate in blood plasma in man and rat. Mevalonate tolerance tests in man..J. Lipid Res. 1979; 20: 716-728Abstract Full Text PDF PubMed Google Scholar), and GC-MS methods (8Hangerfeldt L. Hellstrom K. Blood concentration and turnover of circulating mevalonate in the rat..Life Sci. 1972; 11: 669-676Crossref Scopus (19) Google Scholar, 9Siavoshian S. Simoneau C. Maugeais P. Marks L. Rodary L. Gardette J. Krempf M. Measurement of mevalonic acid in human urine by bench top gas chromatography-mass spectrometry..Clin. Chim. Acta. 1995; 243: 129-136Crossref PubMed Scopus (22) Google Scholar, 10Woollen B.H. Holme P.C. Northway W.J. Martin P.D. Determination of mevalonic acid in human urine as mevalonic acid lactone by gas chromatography-mass spectrometry..J. Chromatogr. B. 2001; 760: 179-184Crossref PubMed Scopus (19) Google Scholar). However, there are very few methods reported for liquid chromatography-tandem mass spectrometry (LC-MS/MS) (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar). The main challenge in developing and validating a method for determining MVA in human plasma was that MVA is a polar, endogenous moiety that circulates in the blood stream at nanogram levels. In most methods, the extraction of MVA from plasma was carried out using ion-exchange resins in the form of mevalonolactone (MVAL) (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar). Complicated procedures such as column switching and gradient flow with long run times were followed (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar). In a modified assay procedure, a polar-end-capped reverse-phase liquid chromatography column was used for the quantification of plasma MVA over a calibration range of 0.5–50 ng/ml in human plasma (12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar). This assay had the advantages of shorter run time and isocratic flow. These methods have reported recovery to be 50–87%. The procedure followed does not capture the effect of any constant impurity/substance that may suppress ionization. The exact recovery can be obtained by comparing the response of processed spiked plasma with that of aqueous samples at the same concentration. The matrix effect can be evaluated by comparing spiked processed plasma blanks with aqueous samples at the same concentration. By knowing the recovery and matrix effect, the sensitivity of the method can be improved. A specific and sufficiently sensitive method was required for the quantification of plasma MVA levels in clinical trials. The reported normal range of human plasma MVA levels is 1.5–11.8 ng/ml (13Naoumova R.P. Marais A.D. Mountney J. Firth J.C. Rendell N.B. Taylor G.W. Thompson G.R. Plasma mevalonic acid, an index of cholesterol synthesis in vivo, and responsiveness to HMG-CoA reductase inhibitors in familial hypercholesterolaemia..Atherosclerosis. 1996; 119: 203-213Abstract Full Text PDF PubMed Scopus (92) Google Scholar). After statin treatment even at the highest dose, the percentage decrease in plasma MVA levels has been shown to be 30–50% (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 14Martin P.D. Mitchell P.D. Schneck D.W. Pharmacodynamic effects and pharmacokinetics of a new HMG-CoA reductase inhibitor, rosuvastatin, after morning and evening administration in healthy volunteers..Br. J. Clin. Pharmacol. 2002; 54: 472-477Crossref PubMed Scopus (151) Google Scholar, 15Lindenthal B. Von Bergmann K. Urinary excretion and serum concentration of mevalonic acid during acute intake of alcohol..Metabolism. 2000; 49: 62-66Abstract Full Text PDF PubMed Scopus (3) Google Scholar). Therefore, the lowest limit of quantification was kept at 0.5 ng/ml and the upper limit was 50 ng/ml. The use of surrogate matrix (water) has been reported (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar) and is necessary to achieve lower levels of quantification. In this study, a simple, robust, and reproducible method has been developed and validated to estimate MVA concentrations in human plasma. Equilibration time for the conversion of MVA to MVAL under acidic pH was further optimized. An approach to differentiate the matrix effect from recovery was performed under validation. The validated method was applied to quantify plasma MVA concentrations in rat to determine the effect of rosuvastatin on plasma MVA levels. MVAL (97%) was obtained from Sigma-Aldrich (Poole, Dorset, UK), and the internal standard (IS), deuterated MVAL (D7-MVAL), was from CDN Isotopes (Pointe-Claire, Quebec, Canada). The cartridges (IST ENV+; 100 mg/3 ml) were procured from International Sorbent Technology (Mid Glamorgan, UK). All solvents and other reagents were of analytical grade. Control human plasma (lithium heparin anticoagulant) for the preparation of quality control (QC) samples was obtained from a blood bank and stored at −70°C before use. The column was a HyPurity Advance column (50 mm × 4.6 mm, 5 μm particle size; Thermo Electron Corp.). The column was kept at ambient temperature. The mobile phase consisted of ammonium formate buffer (10 mM, pH 8.0, adjusted with liquid ammonia) and acetonitrile (70:30, v/v). The flow rate was 0.8 ml/min, and the total run time was 3 min. The liquid chromatograph (Agilent 1100; Agilent Technologies, Inc., Palo Alto, CA) was coupled to a mass spectrometer with a turbo electrospray ion source (4000 Qtrap; Applied Biosystems, Foster City, CA) and was used in negative ionization mode with the following source settings. The turbo ion-spray interface was maintained at 530°C with zero air nebulization. The zero air was kept at a pressure of 70 p.s.i. The turbo ion-spray drying gas (zero air) was kept at a pressure of 70 p.s.i. The collision-activated dissociation gas pressure was 7 p.s.i., and the curtain gas pressure was 30 p.s.i.; turbo ion-spray voltage was −3,500 V. Declustering potential was −35 V; entrance potential was −10 V; collision energy was −20 V; collision cell exit potential was −1 V; and channel electron multiplier was 2,600 V. The multiple reaction monitoring pair monitored was m/z 147 → 59 for MVA and m/z 154 → 59 for D7-MVA, with a dwell time of 200 ms. The autosampler cooler was maintained at 10°C. Analyst software (version 1.4; Applied Biosystems) was used for data registration and calibration. After validation, to study the effect of rosuvastatin on plasma MVA levels in rat (n = 5), the method was used to quantify plasma MVA concentrations after 10 mg/kg oral administration. Blood samples were collected in tubes containing lithium heparin before drug administration and 0.5, 1, 1.5, 6, 16, and 24 h thereafter. The samples were centrifuged at 2,500 g for 10 min. The separated plasma was stored at −70°C until analysis. Samples were thawed in water. Plasma aliquots of 500 μl were added to a glass tubes to which were also added IS (100 μl, 200 ng/ml), 0.1 N HCl (1 ml), and water (0.5 ml); the tubes were than vortex-mixed. The sample solution was allowed to equilibrate for 30 min to convert MVA to MVAL. Each sample solution was individually transferred to a solid-phase extraction cartridge (IST ENV+; 100 mg/3 ml) that had been preconditioned with methanol (1 ml) followed by 0.1 N HCl (1 ml). Each cartridge was washed with 0.1 N HCl (1 ml) followed by water (1 ml) and 15% methanol in water. The cartridges were allowed to dry. The analytes were eluted with 3 × 0.5 ml of methanol. The resulting methanol extract solutions were evaporated to dryness under a stream of nitrogen at 15 p.s.i. and 40°C bath temperature for 15 min. The residues were reconstituted in 0.2% ammonium hydroxide solution (100 μl) to convert MVAL to MVA. Aliquots of 10 μl were injected into the LC-MS/MS apparatus for analysis. The calibration curve (CC) standards were prepared in water by adding known amounts of MVA. Lower limit of quantification (LLOQ) and control samples were obtained by MVA in the concentrations were 0.5 and control and control samples were obtained by in plasma with concentrations of and The and samples were stored at The endogenous MVA obtained in plasma was added to spiked plasma samples to concentrations for and All calibration consisted of sample and calibration in the concentration range of 0.5–50 ng/ml. The concentrations were for and The resulting were the The approach of using water for the preparation of and lower standards has been and reported (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar). A was performed for water and plasma. plasma and water (n = were prepared to the sample preparation procedure and for The recovery was performed at levels by comparing the response of processed samples with of injected The were in 0.2% ammonium hydroxide solution to the study the matrix effect, plasma samples were processed and spiked to and The response was with injected samples at and levels. and and accuracy were evaluated by known amounts of MVA and IS in plasma (n = different concentrations were and samples were prepared to the procedure and accuracy were and accuracy were on and samples were and times with water for analysis was performed at and the processing at and levels. The samples were processed to the procedure in The of MVA was in human plasma and water at temperature for h and in an for The plasma and water samples stored at −70°C solution were performed at temperature for h and in for In a study was in human plasma stored at The of was in an for solution were performed at temperature for h and in for solution were carried out at the and IS was prepared from and The drug and IS response of stored and were In other of and were A liquid mass method for the estimation of MVA in human plasma has been developed and validated to the of The plasma was validated over a concentration range of ng/ml. was by solid-phase extraction using The reconstituted samples were by LC-MS/MS using a HyPurity Advance × 50 The times of MVA and were 0.8 and with a total run time of 3 min. of MVA in and samples are shown in The lower limit of quantitation was 0.5 ng/ml for MVA. The precision and accuracy for MVA at 0.5 ng/ml were and The linearity of the method was by a analysis of an standard The calibration were shown to be from 0.5 to ng/ml. calibration of the of MVA to IS the concentration of calibration standards were by analysis with of The were during the of validation. The of the assay was by the percentage of over the concentration range of and samples during the of validation. The accuracy of the assay was as the of the of the of the samples to as precision from to and accuracy from to precision from to and accuracy from to precision from to and accuracy from to The temperature at h was for MVA and for The solution on the for MVA was and that for IS on the was The that MVA and IS are stable for The from to for MVA and from to for The of MVA in human plasma from to and to for and MVA was to be stable to and the from to MVA was to be stable for to of storage and the from to The recovery of MVA and IS was for spiked samples and of for MVA and for The percentage matrix effect was for and for of MVA and MVA samples in water and samples in MVA from on the same with same samples (n = top on the same with same samples (n = sample on the same with same samples (n = calibration MVA sample of deuterated mevalonic mevalonic quality on the same with same samples (n = in a new of deuterated mevalonic mevalonic quality The for and precision and accuracy for times were and precision and accuracy for times were and These analysis for μl) and μl) analysis and accuracy for were and at and accuracy were and and in a new The validated method was applied to measure MVA in rat plasma. μl) of plasma was used and in water. The samples were run to the Plasma concentrations decreased after oral rosuvastatin (10 administration (Fig. Because the reduction was assay sensitivity (0.5 was In this study, a reverse-phase method with mass using as an IS was of and aqueous were and better chromatography with lower was achieved using ammonium formate buffer (10 mM, pH 8.0) and acetonitrile (70:30, as the mobile was observed in the range of flow rate and was to 0.8 The extraction of MVA is (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar). The sample was with 0.1 HCl to convert MVA to MVAL. The time was to 30 min after response at and min. and solid-phase extraction procedures were for the extraction of MVA from plasma. sample and were obtained using The method is and for the determination of MVA in human plasma at very concentrations over a concentration range to 50 ng/ml. The method has been validated for a of samples a total run time of The use of water as a surrogate matrix the assay to be used to the required lower limit of 0.5 which not be standards were prepared in plasma containing endogenous MVA. In reported methods, recovery was by comparing processed plasma concentrations spiked processed plasma samples (11Abrar M. Martin P.D. Validation and application of an assay for the determination of mevalonic acid in human plasma by liquid chromatography tandem mass spectrometry..J. Chromatogr. B. 2002; 773: 103-111Crossref PubMed Scopus (16) Google Scholar, 12Jemal M. Schuster A. Whigan D.B. Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard..Rapid Commun. Mass Spectrom. 2003; 17: 1723-1734PubMed Google Scholar). This approach to determine recovery does not capture the effect of any constant endogenous that may the the recovery not be the validation, recovery was by comparing processed plasma sample concentrations the same aqueous concentrations for MVA and prepared in solution ammonium The matrix effect was by comparing spiked processed plasma samples the same aqueous concentrations for MVA and A significant matrix effect was observed. The recovery is attributable to the matrix effect. method different plasma samples were spiked to 10 ng/ml to endogenous MVA. The response observed in spiked plasma was increased with that in and were with can be that the of constant endogenous other than MVA any effect during sample processing can to the matrix effect. this method can be used to plasma MVA The method was applied to estimate rat plasma MVA levels after a oral of rosuvastatin (10 the rat is not an for has been used to study the mevalonate (2Popljak G. Boehm C. Parker T.S. Edmond J. Edwards P.A. Fogelman D.A. Determination of' mevalonate in blood plasma in man and rat. Mevalonate tolerance tests in man..J. Lipid Res. 1979; 20: 716-728Abstract Full Text PDF PubMed Google Scholar, L. Hellstrom K. Blood concentration and turnover of circulating mevalonate in the rat..Life Sci. 1972; 11: 669-676Crossref Scopus (19) Google Scholar, H. of plasma mevalonate in and Lipid Res. Full Text PDF PubMed Google Scholar). was in and the normal range of plasma MVA in was to be as reported H. of plasma mevalonate in and Lipid Res. Full Text PDF PubMed Google Scholar). A significant decrease was observed in rat plasma MVA levels after rosuvastatin administration. In a simple, sufficiently and reproducible method was developed for the quantification of MVA in and the developed method has been The that MVA was stable during normal assay procedures and in storage This clinical samples to be stored and The matrix effect and recovery be to the sensitivity of the method and to capture the effect of any endogenous The use of MVA as biomarker to be This was by a by The the of the of and for in this study and for the analysis of The are also to for at the

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