Vol-2, Issue-3, July-2011

ISSN: 0976-7908

Makwana et al

PHARMA SCIENCE MONITOR

AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

BIOANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF ENALAPRIL, ENALAPRILAT AND HYDROCHLOROTHIAZIDE IN HUMAN SERUM BY LC/MS/MS DETECTION
K. Makwana*1, R. Dhamecha2 and N. Pandya3
1 2

Research Scholar, Singhania university, Pacheri Bari(Jhun jhunu),Rajasthan. Department of pharmaceutical technology, M.S.university, Vadodara-390001. 3 Vibgyor clinical research, 45 world business house, parimal garden, ellisbridge, Ahmedabad-380006.

ABSTRACT Enalapril and Enalaprilat and Hydrochlorothiazide were extracted from an aliquot of human serum using solid phase extraction method, and then injected into a liquid chromatograph, equipped with mass spectrometry detector. Internal standard method was used for quantitation of Enalapril, Enalaprilat and Hydrochlorothiazide. Linear regression with 1/X2 weighting was performed to determine the concentration of the drug from serum .Hydroflumethiazide was used as internal standard. A common solid phase extraction procedure for the isolation of drug and its metabolite was developed from serum samples. The samples were analysed on API 3200 Triple quadrapole mass spectrometer using Chromolith, RP-18e column in atmospheric pressure electrospray ionization. The mobile phase composition was a isocratic mixture of 0.01% Ammonia in water : acetonitrile (30:70 %v/v). The method was validated over a linear range of 0.50 500 ng/ml and the limit of quantification were 0.50 ng/ml. Recoveries were observed above 70% for all the three analytes. The storage stability of Quality control samples was investigated under various conditions. Key words: Enalapril, Enalaprilat, Hydrochlorothiazide, LC-MS-MS, Pharmacokinetic studies INTRODUCTION Enalapril N-[(1S)-1-(ethoxycarbonyl)-3phenylpropyl]-L-alanyl-L-proline, is a prodrug. Enalapril is a pro-drug; following oral administration, it is bioactivated by hydrolysis of the ethyl ester to Enalaprilat, which is the active angiotensin converting enzyme inhibitor. Enalapril Maleate is the maleate salt of Enalapril, the ethyl ester of a www.pharmasm.com IC VALUE 4.01 110

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long-acting angiotensin converting enzyme inhibitor, Hydrochlorothiazide is a diuretic and antihypertensive. It is the 3,4-dihydro derivative of Chlorothiazide. Enalaprilat has

been shown to be effective in the treatment of hypertension and congestive heart failure. Various analytical methods for the estimation of Enalapril in the given dosage form were reported in literature which includes high performance liquid chromatography with Ultra violet detection [1] , capillary electrophorosis formation of ternary complex [3]. Recently, HPLC coupled with mass spectroscopic detection has been extensively used for pharmaceutical analysis. For example , A high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using solid phase extraction for the simultaneous determination of plasma concentrations of Enalapril and Enalaprilat in hypertensive patients treated with different pharmaceutical formulations [4]. Along with the advancement in LCMS techniques for estimation of drugs in biological matrix different techniques of extractions were also applied like liquid- liquid extraction
[5] [6] [7] [8] [2]

and flow injection analysis based on the

, protein precipitation

and solid phase extraction

to extract Enalapril and

Enalaprilat from human plasma samples. Hydrochlorothiazide was extracted by solid phase extraction method [9]. From all the extraction procedures solid phase extraction is the best technique for extraction of drug from biological matrix as it possess several advantages like (a) fast , simple and direction sample injection (b) no waste generation as practiced in liquid liquid extraction. (c) the possibility of interfacing with major chromatographical techniques and finally (d) . possibility for obtaining large preconcentration factors and freedom from the contamination and reusability of adsorbants. In this article, we have validated a simple and selective high performance liquid chromatography couples with mass spectrometry method for the detection of Enalapril, Enlaprilat and Hydrochlorothiazide from human serum rather than plasma as per USFDA guidelines [10]. Hydroflumethiazide was used as internal standard. Enalapril, Enalaprilat and Hydrochlorothiazide were extracted from an aliquot of human serum using solid phase extraction method and then injected into a liquid chromatograph, equipped with mass spectrometry detector. Internal standard method was used for quantitation of Enalapril, Enalaprilat and Hydrochlorothiazide. Linear regression www.pharmasm.com IC VALUE 4.01 111

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with 1/X2 weighting was performed to determine the concentration of the drug from serum samples. All regressions and figures presented in this validation report were generated by analyst software version 1.4.1. MATERIALS AND METHODS Enalapril, Enalaprilat, Hydrochlorothiazide and Hydroflumethiazide were purchased from Synfine Research, Canada or Sigma Aldrich, USA. Acetonitrile, methanol, Ammonia,

formic acid and Orthophosphoric acid were obtained from

Qualigens (Worli, Mumbai) India. De-ionized water was prepared on Milliq Laboratory Plant (Millipore, Bedford. USA). Organic solvents and reagents used were of analytical grade. HLB 30mg 1cc solid phase extraction cartridge (Oasis, Waters, USA) was used for sample clean up procedure. Serum was prepared from the blood obtained from suraktam blood bank, Vadodara. Equipments and chromatographic conditions The chromatographic system consist of LC-2010HT (Shimadzu,Japan) equipped with SIL-HTc autosampler. Mass Spectrometric analysis were conducted using API 3200 Q-trap Triple quadrapole instrument (Applied Biosystem, Sciex, Concord, Canada), equipped with a pneumatically assisted APCI(heated nebulizer) and ESI (electrospray) ionization source, which was operated in negative mode. The whole system was controlled using Analyst software version 1.4.1(Applied-Biosystem-Sciex, Concord, Canada). Chromatographic separation was achieved on Merck Chromolith, RP-18e (100*3.0 mm) analytical column maintained at 25C temperature. Mobile phase composition was a mixture of 0.01% Ammonia in 1000 ml deionized water and acetonitrile with ratio of 30:70 %v/v. Flow rate was maintained at 0.7 ml/min. The solid phase extraction cartridge was a hydrophilic-lipophilic balanced copolymer extraction column (1ml, 30mg, Waters Oasis HLB, Waters, USA) MS tuning [10] Tuning of mass spectrometer involves optimizing voltages, currents, flow rates and optimization of ion source parameters to achieve the maximum mass spectral sensitivity and proper resolution.

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Mobile phase was introduced in to the mass spectrometer via the ESI source operating in the negative ion mode under multiple reaction monitoring conditions (MRM). Quantitation was performed using selective ion Monitoring (SIM) mode at m/z 375.07, 347.10, 295.86 and 329.88 for Enalapril, Enalaprilate, Hydrochlorothiazide and Hydroflumethiazide respectively. Preparation of calibration standard Stock solution of Enalapril, Enalaprilat and Hydrochlorothiazide was prepared by accurately weighing and dissolving respective reference standards in methanol to give

the final concentration of 100 g/ml of each. Stock solution of internal standard i.e Hydroflumethiazide was obtained in methanol at a concentration of 500 ng/ml and was used directly for serum sample preparation. Stock solution of Enalapril, Enalaprilat and Hydrochlorothiazide was further diluted with methanol to give serial concentrations of 10.00, 20.00, 40.00, 100.00, 200.00, 1000.00, 2000.00, 4000.00, 10000.00 ng/ml to form working solution of calibration standards. Quality control standard solutions of Enalapril, Enalaprilat and Hydrochlorothiazide were prepared in methanol at concentration of 30.00, 600.00 and 8000.00 ng/ml. Working solution of analytes as well as internal standard were stored at 4C . Sample Preparation A common procedure for the isolation of Enalapril, Enalaprilat and Hydrochlorothiazide from serum samples prior to LC/MS/MS was developed. For

analysis of Enalapril, Enalaprilat and Hydrochlorothiazide, 25 l of Hydroflumethiazide, 500 ng/ml and 475l of 10% aqueous ortho phosphoric acid solution were added to 500 l human serum. The mixture was vortexed for several seconds. Conditioned the HLB cartridge with 1 ml methanol followed by equilibration with 1 ml water. 1 ml of serum sample was loaded, washed with 1 ml of 2% methanol and 1 ml water. Eluted with 525 l of methanol (100%). Method validation [11] The method was validated for specificity, linearity, precision, accuracy, recovery and stability. Specificity

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Specificity was determined by analyzing six different lots to check interference at the retention time of Enalapril, Enalaprilat, Hydrochlorothiazide and Hydroflumethiazide. Linearity Linearity of calibration standards (n=9) for all three analytes were assessed by plotting peak area ratio (y) of analyte to internal standard against the concentration (x) of analytes. The calibration curves were constructed by weighted ( 1/x2) least square linear regression. Precision and Accuracy To determine intraday precision, replicate analysis of quality control sample was performed on the same day. The run consisted of one set of calibration standards and five replicates each of low, middle and high concentration quality control sample. The interday precision was accessed by analysis of batches on different days. Precision was expressed as % CV. Recovery The extraction efficiency of Enlapril, Enalaprilat, Hydrochlorothiazide and Hydroflumethiazide were expressed in term of recovered concentration of analyte and internal standard added to a biological matrix prior to extraction (recovery QC) versus concentration obtained with biological sample where analyte and internal standard were added following extraction. All analysis was performed in triplicate at three analyte concentrations. Percentage drug recovery with corresponding %CV was determined for each serum sample fortified with analyte. Stability The stability of Enalapril, Enalaprilat and Hydrochlorothiazide were tested in serum by short-term stability, long-term stability and freeze-thaw stability. To test the short and long term stability of these analytes in serum, six replicates of each were stored under different conditions. The short and long-term stability tests were performed at room temperature for 24 hours and at -200 C for 35 days. Freeze-thaw stability testing was performed for three frozen and thawed cycles. Freezing was performed at -200 C for 24 hours and thawing at room temperature. The results of freeze-thaw and short and long-term stability were compared with the average of intra-day calibration curves.

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Vol-2, Issue-3, July-2011 RESULTS Limits of Quantitation

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The lower limit of quantitation i.e., lowest standard level with a coefficient of variation less than 20 % was 0.500 ng /ml, for Enalapril , Enalaprilat and Hydrochlorothiazide with between-batch coefficient of variation was 5.59%,4.83% and 1.59% respectively and accuracy was 100.00%, 96.92% and 101.72% respectively. The upper limit of quantitation for Enalapril, Enalaprilat and Hydrochlorothiazide was 500 ng/ml with between-batch coefficient of variation was 2.60%, 2.04 and 3.16 % respectively and accuracy was 105.77%, 102.94% and 101.00% respectively Linearity and Sensitivity Good linearity was achieved over the concentrations in the range of 0.500 to 500 ng/ml for enalapril, enalaprilat and hydrochlorothiazide. The data of linearity are listed in Table 1. The limit of quantification (LOQ) was 0.50 ng/ml using 500 L of serum for enalapril, enalaprilat and hydrochlorothiazide with accuracy, precision 20%. Back calculations were made from the calibration curves to determine Enalapril, Enalaprilat and Hydrochlorothiazide concentration of each calibration standard. The regression equations of these curves and their coefficients were calculated as follows: Enalapril, y = 0.0197x + 0.0010, (R = 0.9976); Enalaprilat, y = 0.0358x + 0.0125, (R = 0.9970); Hydrochlorothiazide, y = 0.0305x + 0.0082(R = 0.9982), Where y is the peak area ratio of analytes to internal standard, x is the concentration of analytes.

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Table No 1: Intermediate precision, accuracy and linear regression parameters of Enalapril, Enalaprilat and Hydrochlorthiazide determination in human serum by LC-MSMS detection. Added Concentration (ng /ml) Enalapril 0.50 1.00 2.00 5.00 10.00 50.00 100.00 200.00 500.00 Calibration curve Slope Intercept Enalaprilat 0.500 1.00 2.00 5.00 10.00 50.00 100.00 200.00 500.00 Calibration curve Slope Intercept Hydrochlorothiazide 0.500 1.00 2.00 5.00 10.00 50.00 100.00 200.00 500.00 Calibration curve Slope Intercept Mean measured Concentration (n = 5)(ng/ml) 0.50 1.01 1.80 4.94 9.38 47.83 104.86 199.64 528.85 0.0197 0.0010 0.48 1.04 2.22 4.91 9.62 47.77 101.28 196.91 514.68 0.0358 0.0125 0.51 0.99 1.91 5.05 9.86 49.06 105.26 200.42 505.00 0.0305 0.0082 Precision (RSD,%) Accuracy (%)a

5.59 12.16 14.86 5.99 3.13 3.93 2.80 2.41 2.60

0.00 1.00 -10.00 -1.20 -6.20 -4.34 4.86 -0.18 5.77

Correlation Coefficient 4.83 9.47 9.77 5.72 5.05 3.54 0.97 5.13 2.04

0.9976 -4.00 4.00 11.00 -1.80 -3.80 -4.46 1.28 -1.55 2.94

Correlation Coefficient 1.59 4.23 10.79 4.89 3.38 2.60 1.64 4.56 3.16

0.9970 2.00 -1.00 -4.50 1.00 -1.40 -1.88 5.26 0.21 1.00

Correlation Coefficient

0.9982

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Vol-2, Issue-3, July-2011 Specificity

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Makwana et al

Presence of any interference from endogenous substances was estimated by analyzing human serum from six different lots of analyte (s) free human serum including hemolised and lipemic serum used for analysis .No significant interference was observed at the retention times of both analyte (s) and internal standard. Figure 1 shows the specificity of blank serum.

Figure 1 A,B,C,D indicates typical chromatogram of blank serum. E, F, G, H indicates typical chromatogram of blank serum spiked with enalapril, enalaprilat, hydrochlorothiazide and hydroflumethiazide.

Precision, Accuracy and Recovery of Method A good precision and accuracy was observed in this method. The intra and interday precision and accuracies are summarized in Table 2 and 3. The intra-day CV (%) were less than 13.25, 10.73 and 13.32 % and inter-day CV (%) were less than 6.99, 7.85 and 13.28 % for Enalapril, Enalaprilat and Hydrochlorothiazide respectively. The intraday accuracies (MRE) were found less than 4.67, 2.67 and -2.61 and the inter-day accuracies were less than 6.99, 7.85 and 13.28 % for Enalapril, Enalaprilat and Hydrochlorothiazide respectively. The recovery of the method was found above 70% for Enalapril, Enalaprilat and Hydrochlorothiazide respectively. These datas were found satisfactory for pharmacokinetic studies.

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Table No 2: Intra- run precision and accuracy for Enalapril, Enalaprilat and Hydrochlorothiazide of QC (n = 5)

Added Concentration (ng/ml) Enalapril

Mean measured Concentration (ng/ml)

standard deviation

CV (%)

Accuracy mean relative error

1.50 1.57 30.00 26.68 400.00 395.09 Enalaprilat 1.50 1.54 30.00 27.31 400.00 402.80 Hydrochlorothiazide 1.50 1.42 30.00 27.05 400.00 389.58

0.21 0.97 18.57 0.17 0.80 13.67 0.19 0.81 10.31

13.25 3.65 4.70 10.73 2.93 3.39 13.32 3.01 2.65

4.67 -11.07 -1.23 2.67 -8.97 0.7 -5.33 -9.83 -2.61

Table No 3: Inter- run precision and accuracy for Enalapril, Enalaprilat and Hydrochlorothiazide of QC (n = 30)

Nominal Concentration (ng/ml) Enalapril

Mean found standard Concentration deviation (ng/ml)

CV (%) Accuracy mean relative error 6.99 5.84 5.21 7.85 6.16 4.85 13.28 4.86 5.04 4.67 4.23 6.33 -1.33 2.97 3.12 2 5.67 3.29

% Recovery

1.50 1.57 30.00 31.27 400.00 425.31 Enalaprilat 1.50 1.48 30.00 30.89 400.00 412.49 Hydrochlorothiazide 1.50 1.53 30.00 31.70 400.00 413.17 Samples Stability

0.11 1.83 22.14 0.12 1.90 20.03 0.20 1.54 20.84

70.44 70.61 70.28 74.80 76.44 73.29 77.58 73.04 70.99

Enalapril, Enalaprilat and Hydrochlorothiazide showed a good stability under the conditions used for storage and processing. The analytes were stable in human serum www.pharmasm.com IC VALUE 4.01 118

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when stored at ambient temperature for atleast 24 hours and showed a good long term stability at -20 C for 35 days. Enalapril, Enalaprilat and Hydrochlorothiazide were

stable under the influence of three freeze/thaw cycles. Stability data of analytes under various storage and freeze thaw conditions were mentioned in Table 4.
Table 4: Stability data of Enalapril, Enalaprilat and Hyrdochlorthiazide Nominal Concentration (ng/ml) Short term stability at about 25 5C for 24 hours. Lower Higher Quality Quality Control control 1.50 400 ng /ml ng /ml found 1.59 0.10 6.48 0.32 422.11 19.59 4.64 -1.50 1.70 0.12 6.93 7.14 425.43 11.88 2.79 0.93 1.56 0.03 2.16 -6.20 404.54 9.35 2.31 -9.94 Long term stability at about -20C 35 days. Lower Higher Quality Quality Control control 1.50 400 ng /ml ng /ml Three freeze/thaw cycles. Lower Quality Control 1.50 ng /ml Higher Quality control 400 ng /ml

Enalapril Mean Concentration (ng /ml )

standard deviation CV(%) % change(bias) Enalaprilat Mean Concentration (ng /ml ) found

1.47 0.08 5.43 0.43

428.59 17.09 3.99 0.94

1.58 0.05 3.02 -1.25

413.78 15.37 3.71 3.61

1.51 0.03 1.77 4.70

406.94 7.74 1.90 -5.11

standard deviation CV(%) % change(bias) Hydrochlorothiazide Mean Concentration (ng /ml) found

1.56 0.10 6.38 -7.24

414.77 22.90 5.52 -2.77

1.59 0.04 2.27 0.82

416.29 12.50 3.00 -0.29

1.37 0.08 5.53 -8.20

379.35 11.38 3.00 -9.60

standard deviation CV(%) % change(bias)

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Vol-2, Issue-3, July-2011 DISCUSSIONS

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A convenient method for the determination of Enalapril, Enalaprilat and Hydrochlorothiazide in human serum has been developed. The analytical method was validated as per the well defined standard operation procedure of Bioanalytical laboratory. The calibration curve for the standard was linear over the range from 0.500 to 500.0 ng/ml . The limit of quantitation (LOQ) was determined to 0.500 ng/ml. ACKNOWLEDGEMENTS I would like to thank Managing Trustee of Parul institute of Pharmacy for providing all financial support for carrying out this research study. REFERENCES 1. Hassan Y, Aboul E, and Laila I: Pharmacokinetic Parameters and Relative
Bioavailability of Two Tablet Formulations of Enalapril Maleate. Instrumentation

Science & Technology 2005; 33, 1,1-8 2. Qin X, Dominic P, and Tsai E: Determination and rotamer separation Enalapril Maleate by capillary electrophoresis. J Chromatogr A 1992; 626, 251258. 3. Ayad M, Shalaby A, Abdellatef H, and Hosny M: Spectrophotometric and AAS
determination of Ramipril and Enalapril through ternary complex formation. J. Pharmac.

Biomed. Anal 2002; 28, 2, 311-321. 4. Lima DM, Mundim IM, Jardim PC, Jardim TS, Diniz DG, and Lima EM: A high performance liquid chromatography-tandem mass spectrometry (LCMS/MS) method using solid phase extraction for the simultaneous determination of plasma concentrations of Enalapril and Enalaprilat in hypertensive patients treated with different pharmaceutical formulations .Ther. Drug Monit 2009; 31, 710-716. 5. Gu Q, Chen X, Zhong D, and Wang Y: Simultaneous determination of Enalapril and Enalaprilat in human plasma by liquid chromatographytandem mass spectrometry .J Chromatogr B 2004; 813, 337-342. 6. Wang P, Liang YJ, and Chen B.M: Simultaneous determination of Enalapril and
Enalaprilat in human plasma by LCMS: application to a bioequivalence study.

Chromatographia 2007; 65, 209.

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7. Lu S, Jiang K, Qin F, Lu X, and Li F: Simultaneous quantification of Enalapril and Enalaprilat in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study. J. Pharmac. Biomed. Anal 2009;1, 163-167. 8. Lee J, Son J, Lee M, Lee KT and Kim DH: Simultaneous quantitation of Enalapril and Enalaprilat in human plasma by 96-well solid-phase extraction and liquid chromatography / tandem mass spectrometry. Rapid Commun. Mass.Spectrom 2003;17,1157-116 9. Parekh SA, Pudage A, Joshi SS, Vaidya VV, Gomes NA and Kamat SS: Simultaneous determination of Hydrochlorothiazide, Quinapril and Quinaprilat in human plasma by liquid chromatography-tandem mass spectrometry. J. Chromatogr .B. Analyt. Technol. Biomed. Life Sci 2008; 873,1,:59-69 10. http://www.astbury.leeeds.ac.uk/facil/MStut/mstutorial.htm 11. US Department of Health and Human Services, Food and Drug Administration, 2001, Guidance for Industry, Bioanalytical Method Validation.

FDA/CDER/CVM, 2001.[Cited 2009 May 2]. http://www.fda.gov/cder/guidance/4252fnl.pdf.

Available from: URL:

For Correspondence:

K. Makwana
Parul institute of pharmacy, waghodia road, waghodia, Vadodara- 391760.

Email: makwana.kunal@gmail.com

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