Liquid Chromatography-Electro Spray Ionization TandemMass Spectrometry for Simultaneous Determination of Amlodipine,

Benazepril and its Active Metabolite Benazeprilat in Human Plasma

M. Farouk 1, Omar Abd Elaziz 1, Shereen M. Tawakkol 2, A. Hemdan 3*, M. Shehata 4

1 Analytical Pharmaceutical Chemistry Department,Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

2 Analytical Pharmaceutical Chemistry Department,Faculty of Pharmacy, Helwan University, Cairo, Egypt

3 Genuine Research Center (GRC) for Bioequivalence Studies, Cairo, Egypt4 Analytical Pharmaceutical Chemistry Department,Faculty of Pharmacy, Cairo University, Cairo, Egypt

Abstract: A selective, sensitive, rapid, and reproducible liquid chromatography- electro spray ionizationtandem mass method has been developed and subsequently validated for the simultaneous determination ofAmlodipine (AML), Benazepril (BENZ), and its active metabolite Benazeprilat (BENZT) in spiked humanplasma, using Moexipril (MOX) as an internal standard (IS). Various modes were tried and the Multiple ReactionMonitoring (MRM) mode was found the most suitable one. The three analytes and Moexipril (IS) were extractedfrom human plasma by simple protein precipitation using acetonitrile as the precipitating solvent. The stationaryphase used was a C18 Sunfire column while water and acetonitrile at 0.1 % formic acid (30:70, v/v) was usedas a mobile phase. The flow rate used was 0.8 mL/min. FDA guidelines were followed for the method validation.The linearity range was found to be 10-200 ng/mL for Amlodipine, 0.5-100 ng/mL for Benazepril and 5-200ng/mL for Benazeprilat and the correlation coefficient was more than 0.9980 for each analyte. The equation ofthe calibration curve was: y = 0.0068x - 0.0072 (r2 = 0.9981, n = 3) for Amlodipine, y = 0.6098x - 0.135 (r2 =0.9988, n = 3) for Benazepril and y = 0.0173x - 0. 0312 (r2 = 0.9987, n = 3) for Benazeprilat. Results foraccuracy and precision showed satisfactory results. Also the method was compared with reported HPLC methodand no significant difference was found.

Key words: Benazepril, Amlodipine, Benazeprilat, LC-tandem mass spectrometry.

IntroductionBenazepril, (3S)-3-[(1S)-1-ethoxycarbonyl-3

phenylpropylamino]-2, 3, 4, 5-tetrahydro-2-oxo-1H-1-benzazepin-1-yl] acetic acid hydrochlorideis an antihypertensive drug, which belongs to thegroup of angiotensin convertase inhibitors. It actson the renin-angiotensin-aldosterone system byinhibition of the conversion of the inactive

angiotensin I to the highly potent vasoconstrictorangiotensin II. It also reduces the degradation ofbradykinin. Benazepril HCl is applied in pharma-cotherapy as a first choice drug for treatment ofarterial hypertension, ischemic heart disease,hypertrophy of the left heart ventricle and postinfarction heart dysfunction 1. In vivo, Benazeprilis hydrolyzed to a pharmacologically active

*Corresponding author (A. Hemdan)E-mail: < hemmdan@yahoo.com > © 2014, Har Krishan Bhalla & Sons

ISSN Print: 2229-7928ISSN Online: 2230-7532

TACL 4 (1) 2014 pp 1 - 13 1

Received 11 October 2013; accepted in revised form 14 December 2013

metabolite, the diacid Benazeprilat, (3-[(1-carbonyl-3-phenyl-(1S)-propyl)-amino]-2,3,4,-5-tetrahydro-2-oxo-1-(3S)-benazepine-1-aceticacid) 2.

Amlodipine is a dihydropyridine calciumchannel blocker 3-4 used in the management ofhypertension and angina pectoris 5. It is reportedthat Benazepril was identified by HPLC 6-9,spectrophotometry 10-16, and with Benazeprilat byliquid chromatography-mass spectrometry 2,17,and gas chromatography-mass spectrometry 18.Also it is reported that Amlodipine wasdetermined by HPLC 19,20, spectrophotometry 21,22.

Benazepril with Amlodipine also weredetermined by HPLC 23, spectrophotometry 24,and liquid chromatography-mass spectrometry 25.Benazepril is used in combination withAmlodipine for the treatment of hypertension, sothere is a need for a sensitive, specific, and repro-ducible method for the simultaneous estimationof Benazepril, its active metabolite Benazeprilat,and Amlodipine.

As to our knowledge, there is no availablereported method for simultaneous determinationof Benazepril, its active metabolite Benazeprilat,and Amlodipine in human plasma. In our studyhere, we tried to develop a sensitive, selectiveand rapid LC-Tandem mass spectrometric methodfor the simultaneous determination of Benazepril,Amlodipine, and Benazeprilat with only oneinternal standard which is MOX.

ExperimentalMaterials and reagents

Amlodipine, Benazepril hydrochloride,Benazeprilat, and the internal standard MOXwere kindly supplied from National OrganizationFor Drug Control and Research (NODCAR,Cairo, Egypt). The purity of the standards werehigher than 99 %. Structures of the compoundsare shown in Fig. 1. HPLC-grade acetonitrile(Sigma-Aldrich), and ultrapure water prepared bya Millipore Milli-Q purification system (Milli-pore Corp. Bedford, MA, USA) were used asmobile phase of the HPLC-MS-MS. Otherreagents were of analytical-grade. Drug-freehuman plasma was obtained from The HoldingCompany for Biological Products & Vaccines

(VACSERA, Cairo, Egypt). Plasma was storedbelow -40°C until used for analysis.

InstrumentsAnalysis was performed on a chromatographic

system Shimadzu LC-20 AT Series HPLC system(Japan) equipped with LC-20 AT HPLC pump,Shimadzu series DGU-20A5 Degasser and aShimadzu SIL-20A autosampler. A chromato-graphic separation was achieved by a Sunfire C18column (50 x 4.6 mm, 5 μm, Waters, USA) and amixture of 0.1 % formic acid-acetonitrile (30:70,v/v) as a mobile phase. The isocratic mobilephase, was filtered through a 0.45 μm membranefilter (Millipore, USA), then sent to sonicator for5 min. The flow rate used was 0.8 mL/min.

Fig. 1. Chemical structures of a) amlodipine,b) benazepril and c) benazeprilat

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 2

The Multiple Reaction Monitoring (MRM) inpositive ion mode was used for quantitativedetermination of the analytes and IS using a MDSSciex (Foster City, CA, USA) API-3200 LC-MS/MS triple quadrupole mass spectrometer, equip-ped with a Turbo ion spray interface at 350°C.The common parameters, collision activateddissociation gas (CAD), nebulizer gas (GS1), andheater gas (GS2) were set at 5, 30, 40 psi,respectively. The compound parameters, collisionexit potential (CXP), collision energy (CE), anddeclustering potential (DP) for the analytes andIS were set at 12, 31, and 56 V respectively.Simultaneous quantitative determination ofAmlodipine, Benazepril, Benazeprilat, and IS wasdone by monitoring the parent / daughter ions atm/z 409.2/238.2, 425.2/351.2, 397/351, and499.4/234.2, respectively. Data acquisition wasmade with Analyst software (version 1.4.2).

Preparation of standard solutions, calibrationstandards and quality control samples

Standard solutions of Benazepril, Benazeprilat,Amlodipine and internal standard (1 mg/mL)were prepared in acetonitrile. Further dilutionwas done for the preparation of standard workingsolution of Amlodipine, Benazepril, Benazeprilat,and MOX (1 μg/mL). The standard workingsolutions were stored at 4°C until used foranalysis, where they were equilibrated to roomtemperature. Different aliquots from the standardworking solutions were used for spiking blankhuman plasma to prepare calibration standardsand quality control samples. Six differentconcentrations of Amlodipine (10-200 ng/mL),Benazepril (0.5-100 ng/mL) and Benazeprilat (5-200 ng/mL) were used as the calibration samples.Quality control samples used were: 10 (LLOQ),15 (LQC), 50 (MQC), 180 ng/mL (HQC) forAmlodipine, 0.5 (LLOQ), 3 (LQC), 30 (MQC),100 ng/mL (HQC) for Benazepril; and 5 (LLOQ),20 (LQC), 50 (MQC), 180 ng/mL (HQC) forBenazeprilat.

Simple protein precipitation A simple protein precipitation was done forextracting Amlodipine, Benazepril, its activemetabolite Benazeprilat, and MOX (IS) from

human plasma. MOX (IS) solution (25 μL of 1000ng/mL) was added to 500 μL plasma. 3 mLacetonitrile was added to precipitate plasmaproteins, and then the samples were vortexed for1 min. The Centrifugation was done at 10,000rpm for 10 min. The clear supernatant wastransferred to a dry test tube and evaporated todryness under a gentle stream of nitrogen gas at40°C, and then the residue was reconstituted with100 μL of the mobile phase, where only 25 μLwas injected for the lc-tandem mass analysis.

ProceduresFor bulk powder drugs

Aliquot volumes from the standard workingsolutions of Amlodipine, Benazepril, and Benaze-prilat were accurately transferred to 10 mLvolumetric flasks to which 500 μL of IS wasadded to provide final concentrations of 10-200ng/mL for Amlodipine, 0.5-100 ng/mL forBenazepril and 5-200 ng/mL for Benazeprilat.The volume was completed to the volume by themobile phase. 25 μL of each concentration wasinjected three times and following the chromato-graphic conditions.

For extraction from human plasmaAliquot volumes from the standard working

solutions of Amlodipine, Benazepril, and Benaze-prilat were added on 500 μL human plasma, then25 μL IS was added on each concentration. Then,3 mL of acetonitrile was added, the samples weremixed on a vortex for 1 min, followed by centri-fugation for 10 min at 10,000 rpm. The clearsupernatant was trans-ferred to a dry test tube andevaporated to dryness under a gentle stream ofnitrogen gas at 40°C, and then the residue wasreconstituted with 100 μL of the mobile phase,where only 25 μL was injected for the lc-tandemmass analysis.

Method validationThe validation of the proposed method was

done as per the FDA guidelines.

SelectivityBlank human plasma from six different sources

were tested for any interference of endogenous

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 3

plasma constituents with the analytes and the IS.

Matrix effectTo test for the effect of the studied matrix on

the quantitative determination of the analytes,extraction was done to blank human plasmasamples as explained earlier, and then spiked withthree different quality control levels with the ISin triplicates. Then the samples were sent foranalysis to obtain the peak areas. The peak areasobtained were compared with those of standardsolutions, where matrix effect is the peak arearatio. Matrix effect is usually done to ensure theprecision and repeatability of the method.

RecoveryCalculation of the recovery % is done to test

for the extraction efficiency of the method. Theextraction procedure was done as mentionedabove for the spiked plasma samples and the peakareas were calculated. Then the peak areas ofsamples spiked after the extraction step wascalculated. The ratio of the two peaks was therecovery %. This was done at three quality controllevels in triplicates.

Calibration curvesSamples used for calibration consist of six

concentrations of Amlodipine (10-200 ng/mL),BENZ (0.5-100 ng/mL), and Benazeprilat (5-200ng/mL). Each concentration was injected threetimes. The relative peak area of each analyte wasrecorded against its concentration, the linearitycurves were constructed and the regressionequations computed.

Precision and accuracyThree different QC levels of each analyte were

analyzed three times intra-daily using theproposed methods under the same experimentalconditions to estimate precision and accuracy.The previous procedures were repeated inter-daily on three different days for the analysis ofthe three chosen concentrations to calculate theinter-assay precision. The relative standard devia-tions were calculated. The criteria for accept-ability of data were accuracy within ±15 % fromthe nominal values and a precision within ±15 %relative standard deviation (RSD), but that of

LQC is not supposed to exceed ±20 % (USDHHS, FDA, CDER, 2001) 26.

StabilityShort-term stability

To test for the short term stability of the threeanalytes, three quality control levels were left for8 hours on bench top at room temperature (bench-top stability). This was done in triplicates.

Freeze and thaw stabilityThree freeze and thaw cycles were applied to

test for the analytes’ stability. The generalprocedure mentioned above was applied for theanalysis. The same criteria of data acceptancementioned under precision apply here.

Stock solution stabilityThe stability of Amlodipine, Benazepril,

Benazeprilat and I.S stock solutions wasperformed by comparison of results from asolution kept for about 60 days at 4°C and from asolution that was extemporaneously prepared. Forthis purpose, three replicates of each analyte wereprepared from each stock solution and injectedin the LC-MS/MS system. The same test wasperformed with the stock solutions of I.S. Resultswere compared to freshly prepared solutions atcorresponding concentration. The samples quali-fied the test if the deviation was within ±15 %.

Results and discussionMass spectrometry

Both positive and negative ion modes were triedfor the simultaneous determination of the mixtureby using only one internal standard. The Positiveion mode was found to be the most suitable forthe LC-tandem mass multiple reaction monitoring(MRM) analyses. Parent ion of each analyte andIS [M + H]+ in the first MS was used to give themost intense product ion peak in MS3 where itwas used for the quantitative measurement.

The parent ion of the protonated Amlodipine,Benazepril and Benazeprilat (m/z 409.2), (m/z425.2) and (m/z 397.0) formed the most intensedaughter ion peak at m/z 238.2, m/z 351.2 and atm/z 351, respectively. Also, the protonated IS(MOX, m/z 499.4) formed the most intensedaughter ion peak at m/z 234.2. Fig. 2.

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 4

Fig. 2. MS Spectrum of a) amlodipine, b) benazepril, c) benazeprilat and d) I.S

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 5

Chromatographic conditionsDifferent ratios of acetonitrile and buffer were

tested for optimum resolution in a reasonable runtime and acceptable peak shape. The mostsatisfactory mobile phase ratio was found to be0.1 % formic acid and acetonitrile (30:70, v/v).Two types of stationary phases C8 and C18 wereinvestigated for the optimum resolution of theanalytes peaks, however the more hydrophobicsunfire C18 was found to be more superior inseparating analytes peaks in a reasonable runtime.

Simple protein precipitationSimple protein precipitation was used to obtain

samples free from endogenous interferents, whichare essential for increasing recovery and mini-mizing matrix effects in LC-tandem massspectrometry. Different precipitating solvents,like methanol, and acetonitrile were tried andtested for removing interferents and obtainingclean plasma samples leading to acceptablematrix effect and recoveries. Acetonitrile provedto be the most suitable, so it was used in theprotein precipitation step.

Selection of ISInternal standard is used to compensate for the

analytes during any step of the analysis. Also,matrix effect could be minimized by choosingsuitable IS. Several compounds were investi-gated, such as torasemid, gliclazide, and MOX,and finally MOX was found to be the mostsuitable IS.

Method validationSelectivity

To test for the method selectivity, blank plasmasamples from six different sources were analyzedto be sure that the endogenous plasma consti-tuents show no interference at the peak region ofthe studied analytes and the IS. The proposedmethod showed no interference at the m/z ratioof the analytes and IS. Fig. 3a, 3b and 3c..

Lower limit of the quantitation (LLOQ)The LLOQ was defined as the lowest concen-

tration on the calibration curves with acceptableprecision and recovery 2. The analyte response atthe LLOQ should be at least five times theresponse of blank baseline 2. To test for LLOQ ofthe proposed method, three replicates of thespiked plasma samples were analyzed at concen-trations of 10, 0.5, 5.0 ng/mL for Amlodipine,Benazepril, and Benazeprilat, respectively. Theprecision and accuracy were found to be 5.09 %and 102 % for AML; 0.97 % and 104 % forBenazepril and 4.29 % and 101 % for Benaze-prilat, respectively.

Recovery and matrix effectDifferent precipitating solvents were tried to

obtain clean samples, minimize matrix effect, andincrease recovery %. Acetonitrile proved to bethe most suitable one for extraction. Therecoveries and matrix effects of Amlodipine,Benazepril, Benazeprilat, and MOX (IS) areshown in table 1. Where three quality controllevels were tested in triplicates

Table 1. The recovery and matrix effect of amlodipine, benazepril, benazepeilat, and IS (n = 3)

Drug C (ng/mL) Recovery ( % ) Matrix Effect ( % )x ± SD RSD % x ± SD RSD %

Amlodipine 15 78.46 ± 4.84 6.17 98.65 ± 2.45 2.4850 79.12 ± 8.64 10.92 99.34 ± 1.48 1.49

180 76.54 ± 2.47 3.23 99.68 ± 1.62 1.63Benazepril 3 82.64 ± 3.87 4.68 99.88 ± 2.16 2.16

30 81.71 ± 5.41 6.62 98.48 ± 3.12 3.17100 81.56 ± 5.23 6.41 98.36 ± 1.57 1.60

Benazeprilat 20 80.69 ± 7.43 9.21 98.24 ± 3.47 3.5350 81.24 ± 4.35 5.35 99.86 ± 2.41 2.41

180 81.34 ± 6.47 7.95 98.63 ± 1.49 1.51IS 10 87.34 ± 4.57 5.23 99.66 ± 2.47 2.48

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 6

Fig. 3a. Blank human plasma showing no interference

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 7

Fig. 3b. Typical MRM chromatograms of a) amlodipine, b) benazepril, c) benazeprilat and d) I.S

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 8

Fig. 3c. MRM chromatograms of Amolodipine, benazepril,benazeprilat and moexipril in spiked human plasam

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 9

LinearityThe three analytes showed linear response

where the correlation coefficients (r2) were all>0.998. The equation of each calibration curvewas: y = 0.0068x - 0.0072 (r2 = 0.9981, n = 3)for AML, y = 0.6098x - 0.135 (r2 = 0.9988, n =3) for Benazepril and y = 0.0173x - 0. 0312 (r2 =0.9987, n = 3) for Benazeprilat.

Precision and accuracyIntra- and inter-day precision and accuracy data

for plasma samples of the three analytes areshown in Table 2. The data showed acceptableresults for both precision and accuracy.

Stability studiesShort term stability

Bench Top stability was checked for eachanalyte by leaving the spiked human plasma for8 hours on bench-top. The concentration of theanalytes never falls outside the accepted rangeas seen in Table 3.

Freeze and thaw stabilityThe stability of the analytes in human plasma

was investigated under the conditions recom-mended by the FDA 26. So freeze-thaw stabilitywas assessed with three sets of stability controlsamples after three freeze-thaw cycles and duringstorage at -80°C up to 30 days. The results areshown in Table 3.

Stock solution stabilityThe stability of the Amlodipine, Benazepril,

Benazeprilat and I.S. stock solutions was per-formed by comparison of results from a solutionkept for about 60 days at 4°C and from a solutionthat was extemporaneously prepared. For thispurpose, three replicates of each analyte wereprepared from each stock solution and injected inthe LC-MS/MS system. The same test wasperformed with the stock solutions of I.S. Resultswere compared to freshly prepared solutions atcorresponding concentration as shown in Table 3.

Statistical AnalysisResults obtained by the proposed method for

the determination of pure samples of Benazepril,Benazeprilat and Amlodipine are statisticallycompared to those obtained by the reportedmethods. The calculated t and F values werefound to be less than their corresponding theoreti-cal ones confirming good accuracy and excellentprecision (Table 4).

ConclusionsFrom the previous discussion, it could be con-

cluded that the described LC-MS/MS methodshow sensitive, specific, reproducible results forsimultaneous determination of Benazepril,Benazeprilat, and Amlodipine in human plasmawith one IS. The methods are suitable for bioequi-valent studies with desired precision and accuracy.

Table 2. The intra-day and inter-day precisions and accuracy ofamlodipine, benazepril and benazeprilat in plasma (n = 3)

C (ng/mL) Intra-day Inter-dayx ± SD Accuracy RSD x ± SD Accuracy RSD

% % % %

Amlodipine 15 14.56±1.41 97.1 9.68 14.78±2.14 98.5 14.4850 51.23±0.57 102.5 1.11 48.89±1.87 97.8 3.82

180 178.63±1.37 99.2 0.77 181.3±0.25 100.7 0.14Benazepril 3 2.87±0.41 95.7 14.29 3.14±0.23 104.7 7.32

30 29.47±1.25 98.2 4.24 31.26±0.54 104.2 1.73100 99.54±0.64 99.5 0.64 99.78±2.54 99.8 2.55

Benazeprilat 20 19.59±0.23 98.0 1.17 20.56±1.84 102.8 8.9550 49.57±1.24 99.1 2.50 49.87±1.57 99.7 3.15

180 177.64±1.71 98.7 0.96 179.9±0.97 99.9 0.54

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 10

Table 4. Statistical comparison for the results obtained by the proposed methods andthe reported methods [6] and [9] for the analysis of BENZ, BENZT, and AML.

Method Drug Mean S.D RSD% N Variance Student’s F testt test(2.18)a (4.81)a

LC/MS BENZ 98.20 1.25 1.270 9 1.562 1.562 1.501BENZT 99.10 1.24 1.251 9 1.538 0.907 3.425AML 99.60 0.57 0.572 9 0.325 1.497 2.603

Reported method [6]b BENZ 99.12 1.02 1.024 6 1.040AML 100.23 0.92 0.905 6 0.846

Reported method [9]c BENZT 99.56 0.67 0.672 6 0.449

a The values in parenthesis are the corresponding theoritical values of t and F at p=0.05.b Separation was achieved on (Inertsil ODS, 250 x 4.6mm, 5μ column) using methanol-acetonitrile-buffer(solution of ammonium acetate and sodium pentanesulphonate ratio, 55:10:35 v/v, PH=3.00 adjusted withphosphoric acid) at a flow rate of 1ml/min, and UV detection at 237 nm using a photodiode array detector.c Separation was achieved on Hypersil BDS C(18) (300 mm x 4.6mm, 5 microm column) using phosphatebuffer (pH 2.6; 10mM) and acetonitrile mixture in a gradient mode as amobile phase, and UV detection at237 nm.

AcknowledgementsThe authors are grateful to Dr A. Hassan, Head

of Genuine Research Centre (GRC). The authors

gratefully acknowledge GRC laboratories fortheir great help to accomplish this work.

Table 3. Stability data of amlodipine, benazepril andbenazeprilat in human plasma at various conditions (n = 3)

C (ng/mL) Bench top ( 8 h ) Three freeze-thaw

Amlodipine 15 x ± SD 14.98 ± 0.43 14.21 ± 3.14Acuuracy % 99.9 94.7

50 x ± SD 50.56 ± 0.48 48.87 ± 2.87Acuuracy % 101.1 97.7

180 x ± SD 180.69 ± 1.24 181.24 ± 3.04Acuuracy % 100.4 100.7

Benazepril 3 x ± SD 3.08 ± 1.12 2.89 ± 2.47Acuuracy % 102.7 96.3

30 x ± SD 30.87 ± 0.76 31.65 ± 2.41Acuuracy % 102.9 105.5

100 x ± SD 99.78 ± 1.42 98.78 ± 0.58Acuuracy % 99.8 98.8

Benazeprilat 20 x ± SD 19.98 0.78 21.12 ± 1.98Acuuracy % 99.9 105.6

50 x ± SD 51.24 ± 1.61 49.47 ± 2.56Acuuracy % 102.5 98.9

180 x ± SD 182.47 0.24 177.69 ± 2.78Acuuracy % 101.4 98.7

M. Farouk et al., / TACL 4 (1) 2014 1 - 13 11

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