methods
Validation
LC-MS/MS method for the quantification of in human plasma and determination of the matrix effect
G.S.Saini,*T.A.Wani,†A.Gautam,*B.Varshney,*T.Ahmed,*K.S.Rajan,*K.K.Pillai,†and J.K.Paliwal 1,*
Department of Metabolism and Pharmacokinetics,*Ranbaxy Rearch Laboratories;and Department of Pharmacology,†Jamia Hamdard,Delhi,India
Abstract A simple,specific,and sufficiently nsitive 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 0.5–50.0ng/ml (r 2.0.99)using deuterated MVA as an internal standard.The lower limit of quantificatio
n was 0.5ng/ml.The assay procedure involved the isolation of MVA from plasma samples using solid-pha extraction.Chromatographic paration was achieved on a HyPurity Advance column with a mobile pha consisting of ammonium formate buffer (10mM,pH 8.0)and acetonitrile (70:30,v/v).Excellent precision and accuracy were obrved.MVA and deuterated mevalonolactone were stable in water and plasma under different storage and processing condi-tions.The recovery obrved was low,which was attribut-able to a significant matrix effect.A significant decrea (30–40%;P ,0.05)was obrved in rat plasma MVA levels after rosuvastatin administration.—Saini,G.S.,T.A.Wani,A.Gautam,B.Varshney,T.Ahmed,K.S.Rajan,K.K.Pillai,and J.K.Paliwal.Validation of the LC-MS/MS method for the quantification of mevalonic acid in human plasma and deter-mination of the matrix effect.J.Lipid Res.2006.47:2340–2345.
Supplementary key words statin .biomarker .liquid chromatography-tandem mass spectrometry
In the biosynthesis of cholesterol,the conversion of HMG-CoA to mevalonic acid (MVA)by HMG-CoA reducta is an early and rate-limiting step (1–3).The statin class of drugs,such as simvastatin,atorvastatin,and rosuvastatin,act on HMG-CoA reducta,resulting in the inhibition of MVA biosynthesis (Fig.1)(4,5).Understanding the reason for incread cholesterol levels and interindividual variability in respon to statin therapy can lead to better
and monitored pharmacotherapy (6).Becau the reduc-tion of MVA levels is an indirect measure of decread cholesterol levels,MVA can be ud 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.The involve enzyme im-munoassay (7),radioimmunoassay (2),and GC-MS meth-ods (8–10).However,there are very few methods reported for liquid chromatography-tandem mass spectrometry (LC-MS/MS)(11,12).
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)(11,12).Complicated procedures such as column switch-ing and gradient flow with long run times were followed (11).In a modified assay procedure,a polar-end-capped rever-pha liquid chromatography column was ud for the quantification of plasma MVA over a calibration range of 0.5–50ng/ml in human plasma (12).This assay had the advantages of shorter run time and isocratic flow.
The 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 respon of procesd spiked plasma with that of aqueous samples at the same concentration.The matrix effect can be evaluated by comparing spiked procesd plasma blanks with aqueous samples at the same concentration.By knowing the recovery and matrix effect,the nsitivity of the method can be improved.
A specific and sufficiently nsitive method was required for the quantification of plasma MVA levels in clinical trials.The reported normal range of human plasma MVA
Manuscript received 15May 2006and in revid form 16June 2006and in re-revid form 21July 2006.
Published,JLR Papers in Press,July 21,2006.DOI 10.1194/jlr.D600018-JLR200
1
To whom correspondence should be addresd.e-mail:jyoti.
Copyright D 2006by the American Society for Biochemistry and Molecular Biology,Inc.
This article is available online at www.jlr
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levels is1.5–11.8ng/ml(13).After statin treatment even at the highest do,the percentage decrea in plasma MVA levels has been shown to be30–50%(11,14,15).There-fore,the lowest limit of quantification was kept at0.5ng/ ml and the upper limit was50ng/ml.The u of surrogate matrix(water)has been reported(11,12)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 con-centrations 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 vali-dated method was applied to quantify plasma MVA con-centrations in rat to determine the effect of rosuvastatin on plasma MVA levels.
EXPERIMENTAL PROCEDURES
Materials
MVAL(97%)was obtained from Sigma-Aldrich(Poole, Dort,UK),and the internal standard(IS),deuterated MVAL (D7-MVAL),was from CDN Isotopes(Pointe-Claire,Quebec, Canada).The cartridges(IST ENV1;100mg/3ml)were procured from International Sorbent Technology(Mid Glamor-gan,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 atÀ70j C before u.
Column liquid chromatography
The column was a HyPurity Advance column(50mm3 4.6mm,5m m particle size;Thermo Electron Corp.).The column was kept at ambient temperature.The mobile pha consisted of ammonium formate buffer(10mM,pH8.0,adjusted with liquid ammonia)and acetonitrile(70:30,v/v).The flow rate was 0.8ml/min,and the total run time was3min.
Mass spectrometry
The liquid chromatograph(Agilent1100;Agilent Technologies, Inc.,Palo Alto,CA)was coupled to a mas
s spectrometer with a turbo electrospray ion source(4000Qtrap;Applied Biosystems, Foster City,CA)and was ud in negative ionization mode with the following source ttings.The turbo ion-spray interface was maintained at530j C with zero air nebulization.The zero air was kept at a pressure of70p.s.i.The turbo ion-spray drying gas(zero air)was kept at a pressure of70p.s.i.The collision-activated dis-sociation gas pressure was7p.s.i.,and the curtain gas pressure was 30p.s.i.;turbo ion-spray voltage wasÀ3,500V.Declustering poten-tial wasÀ35V;entrance potential wasÀ10V;collision energy was À20V;collision cell exit potential wasÀ1V;and channel electron multiplier was2,600V.The multiple reaction monitoring pair monitored was m/z147fi59for MVA and m/z154fi59for D7-MVA,with a dwell time of200ms.The autosampler cooler was maintained at10j C.Analyst software(version 1.4;Applied Biosystems)was ud for data registration and calibration. Sample collection
After validation,to study the effect of rosuvastatin on plasma MVA levels in rat(n55),the method was ud to quantify plasma MVA concentrations after10mg/kg oral administration. Blood samples were collected in tubes containing lithium hepa-rin before drug administration and0.5,1,1.5,6,16,and24h thereafter.The samples were centrifuged at2,500g for10min. The parated plasma was stored atÀ70j C until analysis. Sample preparation
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Samples were thawed in water.Plasma aliquots of500m l were added to a glass tubes to which were also added IS(100m l, 200ng/ml),0.1N HCl(1ml),and water(0.5ml);the tubes were than vortex-mixed.The sample solution was allowed to equili-brate for30min to convert MVA to MVAL.Each sample solution was individually transferred to a solid-pha extraction cartridge (IST ENV1;100mg/3ml)that had been preconditioned with methanol(1ml)followed by0.1N HCl(1ml).Each cartridge was washed with0.1N HCl(1ml)followed by water(1ml)and 15%methanol in water.The cartridges were allowed to dry.The analytes were eluted with330.5ml of methanol.The resulting methanol extract solutions were evaporated to dryness under a stream of nitrogen at15p.s.i.and40j C bath temperature for 15min.The residues were reconstituted in0.2%ammonium hydroxide solution(100m l)to convert MVAL to MVA.Aliquots of10m l were injected into the LC-MS/MS apparatus for analysis. Standard curves
The calibration curve(CC)standards were prepared in water by adding known amounts of MVA.Lower limit of quantification (LLOQ)QC and low-quality control(LQC)samples were obtained by spiking MVA in water;the final concentrations were0.5and 1.9ng/ml,respectively.Middle-quality control(MQC)and high-quality control(HQC)samples were obtained by spiking in plasma with concentrations of21.9and41.3ng/ml,respectively.The bulk-spiked CC and QC samples were stored at
À70j C.The endogenous MVA level obtained in plasma(12.2ng/ml)was added to spiked plasma samples to obtain corrected concentrations for MQC and HQC samples.All calibration curves consisted of one blank sample and eight calibration points in the concentration range of0.5–50ng/ml.The concentrations were corrected for potency and amount weighed.The resulting peak area ratios were plotted against the concentrations.
Validation
Specificity.The approach of using water for the preparation of CC and lower QC standards has been verified and reported previously(11,12).A specificity exerci was performed for both water and plasma.Individual blank plasma samples,LLOQ QC samples,and water(blank)(n56)were prepared according to the sample preparation procedure described above and screened for interference.
Recovery.The recovery exerci was performed at all QC levels by comparing the respon(area)of procesd QC
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with tho of directly injected QC samples.The dilutions were made in0.2%ammonium hydroxide solution to keep conditions the same.
Matrix effect.To study the matrix effect,blank plasma samples were procesd and spiked later to obt
ain MQC and HQC concentrations.The respon(area)was compared with directly injected samples at MQC and HQC levels.
一纳Inter-assay and intra-assay imprecision and accuracy.Inter-assay and intra-assay imprecision and accuracy were evaluated by spiking known amounts of MVA and IS in plasma(n55).Four different concentrations were ud,and samples were prepared according to the procedure mentioned above.Intra-assay imprecision and accuracy were assd within one batch,whereas inter-assay im-precision and accuracy were assd on three parate occasions.
Dilution integrity and partial volume analysis.MQC and HQC samples were diluted two and four times with water for dilution integrity.Partial volume analysis was performed at one-half and one-fourth the processing volumes at MQC and HQC levels.The samples were procesd according to the procedure mentioned above in five replicates.
Stability.The stability of MVA was studied in human plasma and water at room temperature(bench top)for6h and in an autoinjector for20h.The bulk-spiked plasma and water samples stored atÀ70j C underwent three freeze-thaw cycles.Stock solution stability studies were performed at room temperature for4h and in refrigeration for22days.In addition,a long-term (28days)stability study was d
one in human plasma stored at À70j C.The stability of D7-MVA was assd in an autoinjector for20h.Stock solution stability studies were performed at room temperature for4h and in refrigeration for112days.Stock solution stability studies were carried out at the MQC level,and working IS was prepared from fresh and refrigerated stock solutions.The drug and IS respon ratios of stored and fresh stocks were compared.In other stability studies,five replicates of LQC and HQC were analyzed.
RESULTS
A high-performance liquid chromatographic mass spectrometric method for the estimation of MVA in hu-man plasma has been developed and validated accord-ing to the principles of Good Laboratory Practices.The plasma was validated over a concentration range of0.5–48.5ng/ml.Sample cleanup was accomplished by solid-pha extraction using C-18,ENV1cartridges.The reconstituted samples were analyzed by LC-MS/MS using a HyPurity Advance(4.6350mm)column.The retention times of MVA and D7-MVA were between0.8and0.9
min,
Fig.2.Reprentative chromatograms.A:Plasma
extract(endogenous background)without internal
standard.B:Mevalonic acid(MVA)sample pre-
pared in water.C:Spiked human plasma.D7MVA,
deuterated mevalonic acid;HQC,high-quality
control;LLOQ,lower limit of quantification.
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with a total run time of3min.Reprentative chromato-grams of MVA in blank plasma,LLOQ QC,and HQC samples are shown in Fig.2.
The lower limit of quantitation was0.5ng/ml for MVA. The between-run precision and accuracy for MVA at 0.5ng/ml were6%and105%,respectively.The linearity of the method was determined by a weighted least-squares regression analysis of an eight point standard curve.The calibration lines were shown to be linear from0.5to 48.5ng/ml.Best-fit calibration lines of the ratio of MVA to IS peak area versus the concentration of calibration standards were determined by least-squares regression analysis with weighting factors of1/x2.The r2values were consistently.0.99during the cour of validation.
The imprecision of the assay was measured by the percentage coefficient of variation over the concentration range of LLOQ QC,LQC,MQC,and HQC samples during the cour of validation.The accuracy of the assay was defined as the absolute value of the ratio of the calculated mean values of the QC samples to their respective nominal values,expresd as percentages.Within-batch precision ranged from1%to18%,and within-batch accuracy ranged from97%to116%.Intra-assay precision ranged from1% to17%,and intra-assay accuracy ranged from98%to 109%.Inter-assay precision ranged from3%to12%,and
inter-assay accuracy ranged from99%to108%.
The room temperature stock stability at4h was101% for MVA and111%for IS.The refrigerated stock solution stability on the22nd day for MVA was105%,and that for IS on the112th day was100%.The autoinjector stability results demonstrate that MVA and IS are stable for20h. The mean stability ranged from97%to102%for MVA and from98%to102%for IS.The mean stability of MVA in human plasma ranged from97%to102%and92%to98% for one and three freeze-thaw cycles,respectively.During bench-top stability analysis,MVA was found to be stable up to4h,and the mean stability ranged from94%to102%. MVA was found to be stable for up to28days of storage (plasma)belowÀ50j C,and the mean stability ranged from100%to107%(Table1).The absolute recovery of MVA and IS was calculated for replicate spiked QC samples(MQC and HQC).Results indicate overall re-coveries of21%for MVA and21%for IS.The percentage matrix effect was46%for analyte and73%for IS.
The results demonstrate acceptable dilution integrity for two and four times.Within-batch precision and accuracy for two times dilution were5–8%and103–104%,respectively, whereas within-batch precision and accuracy for four times dilution were6–7%and100–107%,respectively.The results demonstrate acceptable partial volume analysis for one-half volume(250m l)and one-fourth volume(1
25m l) analysis(Table2).Within-batch imprecision and accuracy for one-half volume were4–8%and104–105%,respectively, whereas at one-fourth volume,within-batch imprecision and accuracy were6–9%and102–103%,respectively(Table2). The validated method was successfully applied to measure MVA in rat plasma.One-fourth volume(125m l)of plasma was ud and read against CC in water.The QC samples were run to cross-validate the method.Plasma concentrations decread significantly after oral rosuvastatin(10mg/kg) administration(Fig.3).Becau the reduction was30–40%, assay nsitivity(0.5ng/ml)was sufficient.
DISCUSSION
In this study,a rever-pha HPLC method with mass spectrometric detection using D7-MVA as an IS was developed.Various combinations of organic and aqueous phas were tried,and better chromatography with lower baline was achieved using ammonium formate buffer
(10mM,pH8.0)and acetonitrile(70:30,v/v)as the mobile pha.Respon was obrved in the range of0.2–
0.8ml/min flow rate and was optimized to0.8ml/min.
The extraction of MVA is highly pH-specific(11,12).The sample was equilibrated with0.1M HCl to convert MVA to MVAL.The equilibration time was optimized to30min after evaluating respon at15,30,45,and60min.Both liquid-
liquid and solid-pha extraction procedures were assd initially for the extraction of MVA from plasma.Better sample cleanup and reproducibility were obtained using
TABLE1.Stability of MVA and D7MVA(LQC samples in water
and HQC samples in plasma)
Mean MVA Concentration
Storage
Condition Baline QC(CV%)
After Storage
(CV%)
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Difference from
Baline
ng/ml%
MVA
Three freeze-thaw cycles a
LQC2(12)2(4)À10
HQC40(2)41(2)1
Bench top(room temperature)(6h)a
LQC2(12)2(7)À8
HQC40(2)40(2)0.5 Autoinjector,refrigerated extracted sample(20h)a
LQC2(12)2(11)1
HQC40(2)41(1)2
Long term,,À50j C(28days)
LQC Read against fresh
calibration curve
2(12)7
HQC42(7)À0
D7MVA(internal standard)
Autoinjector,refrigerated extracted sample(20h)
LQC13(9)13(8)À1
HQC1(2)1(1)À3
CV,coefficient of variation;D7MVA,deuterated mevalonic acid;
HQC,high-quality control;LQC,low-quality control;MVA,mevalonic
acid;QC,quality control.
a Done on the same day with same baline samples(n55).
TABLE2.Dilution integrity and partial volume analysis
Dilution Integrity Partial Volume Analysis
QC Sample MQC HQC MQC HQC MQC HQC MQC HQC Dilution/
volume
24242424 Imprecision86578946 Accuracy104100104107104102105103 Number55555555 MQC,middle-quality control.
LC-MS/MS method for quantification of MVA2343
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polystyrene-divinylbenzene cartridges.The method de-scribed is nsitive,lective,preci,and accurate for the determination of MVA in human plasma at very low con-centrations(,1ng/ml)over a concentration range extend-ing up to50ng/ml.The method has been validated for a maximum batch size of103samples(with a total run time of6.5h).The u of water as a surrogate matrix allows the assay to be ud down to the required lower limit of 0.5ng/ml,which would not be possible if standards were prepared in plasma containing endogenous MVA.
In previously reported methods,recovery was deter-mined by comparing procesd plasma concentrations against spiked procesd blank plasma samples(11,12). This approach to determine recovery does not capture the effect of any constant endogenous substance that may reduce the respon.Hence,the calculated recovery will not be the actual recovery.During validation,recovery was calculated by comparing procesd plasma QC sample concentrations against the same aqueous concentrations for MVA and D7-MVA prepared in reconstitution solution (0.2%ammonium hydroxide).
The matrix effect was determined by comparing spiked procesd blank plasma QC samples agains
t the same aqueous concentrations for MVA and D7-MVA.A signifi-cant matrix effect was obrved.The low recovery is attributable to the matrix effect.During method develop-ment,six different plasma samples were spiked to add 10ng/ml to endogenous MVA.The respon obrved in spiked plasma was incread proportionately compared with that in blank plasma,and similar results were en with QC samples.It can be concluded that the prence of some constant endogenous substance other than MVA or any reagent effect during sample processing can con-tribute to the matrix effect.Hence,this method can be ud to obtain accurate plasma MVA concentrations. The method was successfully applied to estimate rat plasma MVA levels after a single oral do of rosuvastatin (10mg/kg).Although the rat is not an adequate model for studying lipid metabolism,it has been ud extensively to study the mevalonate pathway(2,8,16).No diurnal rhythm was obrved,unlike in humans,and the normal range of plasma MVA in rats was found to be20–40ng/ml,as re-ported previously(16).A significant decrea was obrved in rat plasma MVA levels after rosuvastatin administration. In conclusion,a simple,sufficiently nsitive,and re-producible method was developed for the quantification of MVA in plasma,and the developed method has been validated.The stability studies demonstrated that MVA was stable during normal assay procedures and in long-term frozen storage conditions(belowÀ50j C).This should allow clinical samples to be stored and analyzed efficiently. The matrix effect and recovery should be differentiated
to improve the nsitivity of the method and to capture the effect of any endogenous interference.The u of MVA as biomarker needs to be explored
further.
This rearch was supported by a grant provided by Ranbaxy Rearch Laboratories(Gurgaon,India).The authors thank the staff of the Department of Metabolism and Pharmacoki-netics for their support in conducting this study and for the analysis of samples.The authors are also grateful to Late Janab Hakeem Abdul Hameed for providing excellent facilities at the university.
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