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SIMULTANEOUS DETERMINATIONOF DOCOSAHEXAENOIC ACID ANDEICOSAPENTAENOIC ACID BY LC-ESI-MS/MS FROM HUMAN PLASMAChinmoy Ghosh a , Vijay Jha a , Chinmay Patra a , Ramesh Ahir a &Bhaswat Chakraborty aa Bio-Analytical Department, Cadila Pharmaceuticals Limited,Dholka, Gujarat, India

Accepted author version posted online: 10 Apr 2012. Version ofrecord first published: 07 Aug 2012

To cite this article: Chinmoy Ghosh, Vijay Jha, Chinmay Patra, Ramesh Ahir & Bhaswat Chakraborty(2012): SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOIC ACID AND EICOSAPENTAENOIC ACIDBY LC-ESI-MS/MS FROM HUMAN PLASMA, Journal of Liquid Chromatography & Related Technologies,35:13, 1812-1825

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SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOICACID AND EICOSAPENTAENOIC ACID BY LC-ESI-MS/MSFROM HUMAN PLASMA

Chinmoy Ghosh, Vijay Jha, Chinmay Patra, Ramesh Ahir, andBhaswat Chakraborty

Bio-Analytical Department, Cadila Pharmaceuticals Limited, Dholka, Gujarat, India

& A sensitive and rapid method based on liquid chromatography=tandem mass spectrometry(LC=MS=MS) with simple single step protein precipitation has been developed and validated forthe quantitative determination of docosahexaenoic acid (DHA) and eicosapentaenoic acid(EPA) in human plasma. After addition of internal standard to human plasma, samples wereextracted by simple protein precipitation using acetonitrile as the precipitating agent. The extractswere analyzed by HPLC with the detection of the analyte in the multiple reaction monitoring(MRM) mode. This method for the simultaneous determination of DHA and EPA is accurateand reproducible, with limits of quantitation of 50.00 ng=mL in plasma. The standard calibrationcurves for both DHA and EPA are linear (r> 0.99) over the concentration ranges 50.00–7498.50 ng=mL in human plasma, respectively. The intra- and inter-day precision over the con-centration range for DHA and EPA are less than 10.16 and 6.72 (relative standard deviation,%RSD), and accuracy is between 91.17–104.74% and 95.81–108.33%, respectively.

Keywords docosahexaenoic acid, eicosapentaenoic acid, LC-MS=MS, matrix effects,poly unsaturated fatty acid, protein precipitation

INTRODUCTION

The x-3 fatty acids found in fish oil, eicosapentaenoic acid (EPA; 20:5n-3) [(5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5, 8, 11, 14, 17-pentenoic acid] anddocosahexaenoic acid (DHA; 22:6 n-3) [(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)-docosa-4,7,10,13,16,19-hexaenoic acid], are essential for growth and devel-opment, and may also play an important role in the prevention and treat-ment of cardiovascular disease, inflammatory diseases, and cancer.[1,2]

Several species of marine fish offer rich dietary sources of polyunsaturatedfatty acids (PUFA), for example EPA and DHA, but these foods are not

Address correspondence to Mr. Chinmoy Ghosh, Research Scientist, Cadila PharmaceuticalsLimited, 1389, Trasad Road, Dholka, Gujarat, India. E-mail: [email protected]

Journal of Liquid Chromatography & Related Technologies, 35:1812–1825, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.627603

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regularly included in the western diet. For the majority of the population,the alternative dietary source of long chain of x-3 fatty acids might be theprecursor, a-linolenic acid (ALA; 18:3 n-3). Previous reports suggested thatincreased intake of ALA, similar to intake of EPA and DHA, may have ben-eficial effects in health and in the control of chronic diseases.[1]

Very few bioavailability studies have been undertaken for EPA and DHA;among them this is the only method that was developed by using LC-MS=MS.Methods currently used for the analysis of mono- and poly-hydroxy fatty acidsinclude gas chromatography (GC)[3–5] andHPLC with a chemiluminescencelabeling method.[6] Among all these published methods, there is only onemethod available for determination of EPA and DHA from human plasma,[5]

and the rest are either from fish,[3] perilla oil,[4] or human serum.[6]

In contrast to all reported methods for simultaneous determination ofEPA and DHA, the current method is the simplest, fastest and most sensitiveone. The other reported methods have very long extraction procedures[3–6]

with comparatively high LLOQ value. The extraction method adopted byChauke et al.[3] used mechanical homogenization followed by liquid-liquidextraction (LLE) andmethyl esterification for GC analysis. In anothermethodreported by Kurowska et al.,[4] several intermediate steps were performed thatincluded liquid-liquid extraction (LLE) followed by hydrolyzed incubation,precipitation, again incubation, and finally LLE followed by GC analysis.Similarly, Rusca et al.[5] performed the LLE, followed by de-esterification,methyl esterification, and again LLE before injecting into GC. LLE followedby chemiluminescence labeling reaction was reported by Hidetaka et al.[6]

Whereas, the present manuscript describes a sensitive, simple, single step pro-tein precipitation technique that achieves a LLOQ value of 50ng=mL. More-over, this is the fastest method with respect to any other reportedmethods,[3–6]

with only 2.25min of analysis time and a very wide range of assay linearity, overthe concentration range of 50.00–7498.50ng=mL. LC-MS=MS combines theresolving power of liquid chromatography with the detection specificity ofMS=MS and overcomes the limitations of the conventionally used approaches,thus providing the means of a rapid, versatile, and sensitive methodology overthe GC methods, which is not widely used for bioanalysis nowadays. Moreover,no methods were reported with as short an analysis time, as simple an extrac-tion technique, or as wide a linearity range. As a result, this developed methodis worthwhile for bioanalysis of EPA and DHA by LC-MS=MS.

EXPERIMENTAL

Chemicals and Reagents

DHA (98% purity) and EPA (99% purity) were obtained from the SigmaAldrich, MO, USA and Nevirapin (internal standard, IS, 100.3% purity) was

Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1813

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purchased from Sequent Scientific Limited, New Mangalore, India.Methanol and acetonitrile (J.T. Baker, IL, USA) was of HPLC-grade, andother chemicals used were of analytical grade. Water used for the prep-aration of the mobile phase and other solutions was collected from MilliQPS (Millipore, NY, USA). Human K2EDTA Plasma, lipemic, and hemolyzedplasma were used during validation and study sample analysis was suppliedby the clinical unit of Cadila Pharmaceuticals Limited, Ahmedabad, India.Plasma was stored at �30� 5�C before sample preparation and analysis.Plasma was obtained by centrifugation of blood treated with the anticoagu-lant K2EDTA and stored at �30�C until analysis.

Instrumentation

The HPLC system with an auto sampler was a Shimadzu LC-20AD(Shimadzu, Tokyo, Japan) coupled with Applied Biosystem Sciex (MDSSciex, ON, Canada) API 4000 Tandem mass spectrometry. The auto sam-pler was SIL-HTC from Shimadzu, Tokyo, Japan. The solvent delivery mod-ule was LC-20AD from Shimadzu, Tokyo, Japan. The chromatographicintegration was performed by Analyst software 1.4.2 (Applied Biosystems,ON, Canada).

Standards and Quality Control Samples

Stock solutions of DHA and EPA were prepared by dissolving the accu-rately weighed reference compounds in methanol to give a final concen-tration of 1000mg=mL of both. The solution was then diluted with diluent(methanol:water 70=30, v=v) to achieve mixed standard working solutionsat concentrations of 50.00, 100.00, 499.90, 1999.60, 3499.30, 4999.00,6198.75, and 7498.50ng=mL for both DHA and EPA. Stock solution of ISwas prepared in methanol at the concentration of 1000mg=mL and dilutedto 30mg=mL with diluent. Structural formulae of DHA, EPA, and IS areshown in Figure 1. All solutions were stored at �30�C and were broughtto room temperature before use.

For the preparation of standard curves or quality control samples, thepre-mixed standard working solutions (10mL) were used to spike blankplasma samples (190mL), both in pre study validation and during the analy-sis of samples from the pharmacokinetic study.

Sample Preparation

To a 0.2mL aliquot of plasma sample, 20mL of internal standard(30 mg=mL) was added. The samples were briefly mixed and 1mL of

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acetonitrile was added. The mixture was vortex-mixed for approximately1min and placed on a centrifuge machine. After centrifugation at4500 rpm for 5min, the upper organic layer was removed and transferredinto HPLC vial and was injected onto the LC–MS=MS system for analysis.

Chromatographic Conditions

Chromatography was performed on a Kromasil-100 C18 analyticalcolumn (50mm� 4.6mm i.d., 5m, Eka Chemicals, Bohus, Sweden). Thecolumn was maintained at 30�C. The compounds were eluted isocraticallyat a flow rate of 0.45mL=min. The mobile phase consisted of 0.1% v=vammonia solution:acetonitrile:methanol (10:81:09, v=v=v).

Mass Spectrometric Conditions

The mass spectrometer was operated in the negative ion detectionmode. Nitrogen was used as the nebulizing, turbo spray, and curtain gas,with the optimum values set at 25, 30, and 25psi, respectively. The tempera-ture of the vaporizer was set at 400�C and ESI needle voltage was adjustedto �4500V. The declustering potential was set at �80, �85, and �80V forDHA, EPA, and IS, respectively. Identification was performed using mul-tiple reaction monitoring (MRM) of the transitions of m=z 327.30!m=z283.20 for DHA, m=z 301.30!m=z 257.20 for EPA and m=z 265.00!m=z182.20 for nevirapine (IS), respectively, with a dwell time of 200 ms pertransition. For collision-induced dissociation (CID), nitrogen was used as

FIGURE 1 Structure of (A) docosahexaenoic acid, (B) eicosapentaenoic acid, and (C) nevirapine(internal standard).


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the collision gas at a pressure of 8 psi. The collision energy was �20V,�22V, and �35V for DHA, EPA, and IS, respectively.

Method Validation

EPA and DHA, are the endogenous compounds, so the back-calculatedconcentration and accuracy were calculated by using a validated MicrosoftExcel sheet, which was validated by SAS software. Four zero standard sam-ples were injected with each calibration curve to calculate the average arearatio of the endogenous components. The calculated average area ratio wassubtracted from the area ratio of each sample to get the actual area ratio ofthe components, which were further used to back calculate the concen-tration and accuracy of calibration standards and quality control samplesusing the validated Excel.

The sample concentrations were calculated using weighted (1=x2) leastsquares linear regression. To evaluate linearity, plasma calibration curveswere prepared and were assayed on three separate days. Accuracy andprecision were also assessed by determining QC samples at three concen-tration levels on three different validation days. The accuracy was expressedby, (observed concentration=spiked concentration)� 100% and the pre-cision by relative standard deviation (RSD). The extraction recoveries ofDHA and EPA at three QC levels were evaluated by comparing peak areasof analytes obtained from plasma samples with the analytes spiked beforeextraction to those spiked after the extraction. The matrix effect was car-ried out by extracting low and high quality control samples in triplicatefrom six different plasma sources. The hemolysis effect and lipemic effectexperiments were carried out at two different concentration levels.

The stability of DHA and EPA in the diluents was assessed by placing QCsamples under ambient conditions for at least 6 hr. The freeze–thaw stabilityof DHA and EPA was also assessed by analyzing QC samples undergoing fourfreeze (�20�C) and thaw (room temperature) cycles. Similarly stability ofDHA and EPA was assessed inside the auto sampler and in plasma at roomtemperature. Subsequently, the DHA and EPA concentrations weremeasured and compared with freshly prepared samples, respectively.

Clinical Protocol

The pharmacokinetic study protocol presented in this paper wasapproved by the Independent Medical Ethics Committee of CadilaContract Research Organization, Ahmedabad, Gujarat, India. In both per-iods, the subject was administered a single dose of omega-3 fatty acid cap-sule along with 200mL of drinking water after an overnight fasting of at


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least 10 hr in each period. The test capsule contains 30mg DHA and250mg EPA, whereas the reference capsule contains 250mg DHA and350mg EPA. The administered subject was a healthy, adult, male, humanvolunteer of Indian origin. In each period, a total of 17 blood samples werecollected including four pre-dose sample prior to drug administration. Theblood samples were immediately centrifuged at 2000 rpm for 10min at 4�C,and the plasma samples were stored at �30�C until LC-MS=MS analysis.

RESULTS AND DISCUSSION

Optimization of Chromatographic Condition

The successful analysis of the analytes in biological fluids using HPLC-MS=MS relies on the optimization of chromatographic conditions, sample prep-aration techniques, chromatographic separation, post column detection,and so forth.[7–10] Thus, for better selectivity and sensitivity, different typesof columns and mobile phases were used. Length of the column varied from50mm to 150mm, and the particle size varied from 3.5m to 5m. Columns ofdifferent types of stationary phase such as C8 and C18 were used which showedsome remarkable effect on peak shape. Finally, Kromasil-100 C18 analyticalcolumn (50mm� 4.6mm i.d., 5m, particle size) was selected for analysis.

The influence of buffer molarities, pH, and types of organic modifier onthe signal intensities was also studied. Initially, 90% acetonitrile: 10% of 0.1%ammonia solution (v=v) at a flow rate of 0.500mL=min was tried but it led toimproper resolution between the other endogenous peaks present in theextracted plasma samples. Therefore, methanol was introduced in a smallquantity, that is, 9% v=v by decreasing the equal volume of acetonitrile.Then, based on the peak intensity and peak resolution, 0.1% ammonia sol-ution (v=v), acetonitrile andmethanol (10:81:09, v=v) as the mobile phase ata flow rate of 0.450mL=min were selected for further studies.

In sample extraction technique, protein precipitation was adopted.Initially, plasma samples were precipitated by using methanol as the preci-pitating agent, but it extracts more endogenous compounds that interferewith the main peak. Then, acetonitrile was used as the precipitating agentwhich produced a better result. Initially, a conventional 2:1(v=v) ratio ofacetonitrile to plasma was used, but due to high extraction recovery, theresponses of the analytes caused the saturation of the detector; as a result,to make a diluted sample, 5:1 (v=v) ratio was selected.

Method Validation

The validation parameters were linearity, sensitivity, accuracy, precision,matrix effects of the assay, and recovery and stability in human plasma,


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according to the U.S. Food and Drug Administration (FDA) guidance forthe validation of Bioanalytical methods.[11]

LinearityLinearity of calibration standards was assessed by subjecting the spiked

concentrations and the respective peak areas using 1=X2 linear least-squaresregression analysis. Linearity ranged from 50.00 to 7498.50 ng=mL for DHAand EPA both (r> 0.990). In aqueous solution, accuracy of all calibrationstandards was within 85–115%, except LLOQ where it was 80–120%.

Specificity and SelectivityAs DHA and EPA are endogenous components, the specificity experiment

was performed for IS only. Six different lots of normal plasma and one lot oflipemic plasma and hemolyzed plasma were analyzed to ensure that noendogenous interference took place with themass transitions of the IS. LLOQlevel samples along with plasma blank from the respective plasma lot were pre-pared and analyzed. In each plasma blank, the response at the retention timeof IS was <5% of the IS response in the respective LLOQ. Figure 2A and 2Brepresents the plasma blank of DHA and EPA, respectively, with IS.

Accuracy and PrecisionFor the validation of the assay, QC samples were prepared at three con-

centration levels of low, medium, and high. Six replicates of each QC sam-ples were analyzed together with a set of calibration standard. The accuracyof each sample preparation was determined by injection of calibration sam-ples and three QC samples in six replicates for 3 d. Obtained accuracy andprecision (inter- and intra-day) are presented in Table 1 for EPA and DHA.The results show that the analytical method is accurate, as the accuracy iswithin the acceptable limits of 100� 20% of the theoretical value at LLOQand 100� 15% at all other concentration levels. The precision around themean value was never greater than 15% at any of the concentration studied.

Limit of QuantitationProcess and inject six LLOQ and six ULOQ samples along with cali-

bration standards in the same range used for calculation of precision andaccuracy.

For DHA, the %CV at LLOQ level was 7.14 and at ULOQ level was 3.69.The average %accuracy at LLOQ level was102.02 and at ULOQ level was101.32. For EPA the %CV at LLOQ level was 4.98 and at ULOQ level was3.74. The average %accuracy at LLOQ level was100.34 and at ULOQ levelwas 99.30.


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Recovery StudyA recovery study was performed by comparing processed QC samples of

three different levels in six replicate with aqueous samples of same level.The recovery of DHA at low quality control (LQC) level was 58.64%,

FIGURE 2 A) Representative chromatogram of plasma blank of DHA and IS and B) Representativechromatogram of plasma blank of EPA and IS. (Color figure available online.)


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medium quality control (MQC) level was 47.96%, and for high qualitycontrol (HQC) level was 49.60%. The mean recovery of DHA was52.07%. % coefficients of variation (%CV) of mean recovery of all threeQCs were 11.04.

The recovery of EPA at low quality control (LQC) level was 46.35%,medium quality control (MQC) level was 41.25%, and for high quality con-trol (HQC) level was 43.88%. The mean recovery of EPA was 43.83%. %CVof mean recovery of all three QCs was 5.82. Recovery of internal standardwas 42.02%.

Matrix EffectsMatrix effect evaluated by eighteen LQC and eighteen HQC samples;

three each from six different plasma lots were processed and analyzed.For DHA the average % accuracy for all LQC level was 97.83 and %CV ofall LQC samples was 3.14 and the average % accuracy for all HQC levelwas 100.56 and %CV of all HQC samples was 2.06. Whereas, for EPA, theaverage % accuracy for all LQC level was 108.93 and %CV of all LQC sam-ples was 2.06 and the average % accuracy for all HQC level was 99.82 and%CV of all HQC samples was 2.05.

Hemolysis EffectsTo determine hemolysis effects, six QC samples were prepared in hemo-

lyzed plasma with three concentration levels of low, medium, and high. Sixreplicates of each QC samples were analyzed together with a set of cali-bration standard prepared in normal plasma. The accuracy of each samplepreparation was determined by injection of calibration samples and threeQC samples in six replicate. For DHA the average % accuracy for LQC levelwas 99.45, for MQC level was 113.32, and for HQC level was 108.83. The%CV of LQC was 7.32, for MQC was 3.72, and for HQC was 3.69.

TABLE 1 Inter-Day and Intra-Day Accuracy and Precision

DHA EPA

Days QC Level Accuracy Precision Accuracy Precision

Day 1 LQC 92.35 7.97 98.42 5.25MQC 108.10 2.05 104.61 4.02HQC 94.35 4.21 96.84 5.03

Day 2 LQC 91.45 2.36 94.81 5.79MQC 99.69 3.68 99.52 2.50HQC 97.27 3.95 97.94 4.47

Day 3 LQC 97.71 5.77 100.05 0.92MQC 104.74 0.93 108.33 1.00HQC 97.07 2.41 99.35 4.02


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For EPA the average % accuracy for LQC level was 105.31, for MQClevel was 111.02, and for HQC level was 103.91. The %CV of LQC was4.85, for MQC was 1.50, and for HQC was 2.02.

Lipemic EffectsTo determine lipemic effects, six QC samples were prepared in lipe-

mic plasma with three concentration levels of low, medium, and high.Six replicates of each QC samples were analyzed together with a set ofcalibration standard prepared in normal plasma. The accuracy of eachsample preparation was determined by injection of calibration samplesand three QC samples in six replicate. For DHA the average % accuracyfor LQC level was 98.73, for MQC level was 106.72, and for HQC levelwas 105.67. The %CV of LQC was 9.03, for MQC was 2.49, and forHQC was 1.97.

For EPA the average % accuracy for LQC level was 93.17, for MQC levelwas 107.34, and for HQC level was 103.80. The %CV of LQC was 3.11, forMQC was 2.65, and for HQC was 2.53.

Stability StudiesThe stability of DHA and EPA were investigated in the stock and working

solutions, in plasma during storage, during processing, after four freeze–thaw cycles, and in the final extract. Stability samples were compared with

TABLE 2 Summary of Stability Data of DHA

Stability QC LevelMean Precision

(%CV)Mean

AccuracyPercentChange

StabilityDuration

Bench top LQC 4.85 85.72 �9.18 8HrHQC 4.09 102.88 5.98

Freeze thaw LQC 5.76 92.70 1.04 4 CyclesHQC 2.01 90.09 2.98

Auto sampler LQC 7.28 97.50 �0.57 24HrHQC 1.86 97.27 �0.47

TABLE 3 Summary of Stability Data of EPA

Stability QC LevelMean Precision

(%CV)Mean

AccuracyPercentChange

StabilityDuration

Bench top LQC 1.42 100.36 0.32 8HrHQC 3.54 106.41 7.11

Freeze thaw LQC 2.98 98.30 2.36 4 CyclesHQC 1.57 91.33 3.26

Auto sampler LQC 4.79 110.39 1.47 24HrHQC 2.31 95.74 �0.82


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freshly processed calibration standards and QC samples. Analytes wereconsidered stable when the change of concentration is �10% with respectto their initial concentration.

The %CV of DHA at LQC and HQC levels for, bench top stability, autosampler stability, and freeze-thaw stability were 4.85, 7.82, and 5.76 and

FIGURE 3 (A) Representative chromatogram of real sample of DHA and (B) Representative chroma-togram of real sample of EPA. (Color figure available online.)


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4.09, 1.86, and 2.01, respectively, whereas, the %CV of PHA at LQC andHQC levels were 1.42, 4.79, and 2.98 and 3.54, 2.31, and 1.57, respectively.

Summary of stability data are presented in Tables 2 and 3 for DHA andEPA, respectively.

Calibration Curve ParametersThe summary of calibration curve parameters was as follows. For DHA

the mean slope and y-intercepts were 0.00573 (Range: 0.0004 to 0.0007)and 0.051282 (Range: �0.0984 to 0.2982), respectively. The mean corre-lation coefficient, r was 0.9985 (Range: 0.9965 to 0.9999).

FIGURE 4 (A) Plasma concentration-time curves for DHA (n¼ 1) and (B) Plasma concentration-timecurves for EPA (n¼ 1). (Color figure available online.)


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For EPA the mean slope and y-intercepts were 0.000618 (Range: 0.0005to 0.0008) and 0.0143 (Range: �0.0629 to 0.0743), respectively. The meancorrelation coefficient, r was 0.9984 (Range: 0.9962 to 0.9999).

APPLICATION

The validated method was applied to determine the concentrationtime profile, following single dose oral administration of capsule inhealthy human volunteer. This developed method can also be appliedfor estimation of EPA and DHA from urine, serum, and other biologicalmatrices to some extent. This method will be a valuable tool during in vitrostudy of EPA and DHA. The method can also be helpful for routineanalysis of EPA and DHA in quality control departments. The chromato-grams obtained from the analysis of real samples are presented inFigure 3A and 3B for DHA and EPA, respectively. After LC-MS=MS analy-sis, the plasma concentration of DHA and EPA were measured. Figure 4Aand 4B shows the plasma concentration-time curves for DHA and EPA,respectively.

CONCLUSION

A LC=MS=MS method with simple protein precipitation has beendeveloped for the simultaneous quantitative determination of DHA andEPA in human plasma. The present method affords the rapid, sensitivity,accuracy, and precision necessary for fast quantitative measurements inpharmacokinetic studies and therapeutic monitoring of DHA and EPA.

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EPA DHA jlcrt

Health & Medicine

Transcript of EPA DHA jlcrt