This article was downloaded by: [Chinmoy Ghosh]On: 09 August 2012, At: 00:20Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Liquid Chromatography & Related Technologies Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljlc20 SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOIC ACID AND EICOSAPENTAENOIC ACID BY LC-ESI-MS/ MS FROM HUMAN PLASMA a a a a Chinmoy Ghosh , Vijay Jha , Chinmay Patra , Ramesh Ahir & a Bhaswat Chakraborty a Bio-Analytical Department, Cadila Pharmaceuticals Limited, Dholka, Gujarat, India Accepted author version posted online: 10 Apr 2012. Version of record first published: 07 Aug 2012To 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, 1812-1825To link to this article: http://dx.doi.org/10.1080/10826076.2011.627603PLEASE SCROLL DOWN FOR ARTICLEFull terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditionsThis article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae, and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.
Journal of Liquid Chromatography & Related Technologies, 35:1812–1825, 2012 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6076 print/1520-572X online DOI: 10.1080/10826076.2011.627603 SIMULTANEOUS DETERMINATION OF DOCOSAHEXAENOIC ACID AND EICOSAPENTAENOIC ACID BY LC-ESI-MS/MS FROM HUMAN PLASMA Chinmoy Ghosh, Vijay Jha, Chinmay Patra, Ramesh Ahir, and Bhaswat ChakrabortyDownloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 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 for the quantitative determination of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human plasma. After addition of internal standard to human plasma, samples were extracted by simple protein precipitation using acetonitrile as the precipitating agent. The extracts were 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 accurate and reproducible, with limits of quantitation of 50.00 ng=mL in plasma. The standard calibration curves 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; 20:5 n-3) [(5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5, 8, 11, 14, 17-pentenoic acid] and docosahexaenoic 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 polyunsaturated fatty acids (PUFA), for example EPA and DHA, but these foods are not Address correspondence to Mr. Chinmoy Ghosh, Research Scientist, Cadila Pharmaceuticals Limited, 1389, Trasad Road, Dholka, Gujarat, India. E-mail: firstname.lastname@example.org
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1813 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 the precursor, a-linolenic acid (ALA; 18:3 n-3). Previous reports suggested that increased intake of ALA, similar to intake of EPA and DHA, may have ben- eficial effects in health and in the control of chronic diseases. 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 acids include gas chromatography (GC)[3–5] and HPLC with a chemiluminescence labeling method. Among all these published methods, there is only one method available for determination of EPA and DHA from human plasma, and the rest are either from fish, perilla oil, or human serum. In contrast to all reported methods for simultaneous determination ofDownloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 EPA and DHA, the current method is the simplest, fastest and most sensitive one. The other reported methods have very long extraction procedures[3–6] with comparatively high LLOQ value. The extraction method adopted by Chauke et al. used mechanical homogenization followed by liquid-liquid extraction (LLE) and methyl esterification for GC analysis. In another method reported by Kurowska et al., several intermediate steps were performed that included liquid-liquid extraction (LLE) followed by hydrolyzed incubation, precipitation, again incubation, and finally LLE followed by GC analysis. Similarly, Rusca et al. performed the LLE, followed by de-esterification, methyl esterification, and again LLE before injecting into GC. LLE followed by chemiluminescence labeling reaction was reported by Hidetaka et al. Whereas, the present manuscript describes a sensitive, simple, single step pro- tein precipitation technique that achieves a LLOQ value of 50 ng=mL. More- over, this is the fastest method with respect to any other reported methods,[3–6] with only 2.25 min of analysis time and a very wide range of assay linearity, over the concentration range of 50.00–7498.50 ng=mL. LC-MS=MS combines the resolving power of liquid chromatography with the detection specificity of MS=MS and overcomes the limitations of the conventionally used approaches, thus providing the means of a rapid, versatile, and sensitive methodology over the 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 method is 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 Sigma Aldrich, MO, USA and Nevirapin (internal standard, IS, 100.3% purity) was
1814 C. Ghosh et al. purchased from Sequent Scientific Limited, New Mangalore, India. Methanol and acetonitrile (J.T. Baker, IL, USA) was of HPLC-grade, and other chemicals used were of analytical grade. Water used for the prep- aration of the mobile phase and other solutions was collected from Milli QPS (Millipore, NY, USA). Human K2EDTA Plasma, lipemic, and hemolyzed plasma were used during validation and study sample analysis was supplied by the clinical unit of Cadila Pharmaceuticals Limited, Ahmedabad, India. Plasma was stored at À30 Æ 5 C before sample preparation and analysis. Plasma was obtained by centrifugation of blood treated with the anticoagu- lant K2EDTA and stored at À30 C until analysis. InstrumentationDownloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 The HPLC system with an auto sampler was a Shimadzu LC-20AD (Shimadzu, Tokyo, Japan) coupled with Applied Biosystem Sciex (MDS Sciex, 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 chromatographic integration 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 1000 mg=mL of both. The solution was then diluted with diluent (methanol:water 70=30, v=v) to achieve mixed standard working solutions at concentrations of 50.00, 100.00, 499.90, 1999.60, 3499.30, 4999.00, 6198.75, and 7498.50 ng=mL for both DHA and EPA. Stock solution of IS was prepared in methanol at the concentration of 1000 mg=mL and diluted to 30 mg=mL with diluent. Structural formulae of DHA, EPA, and IS are shown in Figure 1. All solutions were stored at À30 C and were brought to room temperature before use. For the preparation of standard curves or quality control samples, the pre-mixed standard working solutions (10 mL) were used to spike blank plasma samples (190 mL), both in pre study validation and during the analy- sis of samples from the pharmacokinetic study. Sample Preparation To a 0.2 mL aliquot of plasma sample, 20 mL of internal standard (30 mg=mL) was added. The samples were briefly mixed and 1 mL of
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1815Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 FIGURE 1 Structure of (A) docosahexaenoic acid, (B) eicosapentaenoic acid, and (C) nevirapine (internal standard). acetonitrile was added. The mixture was vortex-mixed for approximately 1 min and placed on a centrifuge machine. After centrifugation at 4500 rpm for 5 min, the upper organic layer was removed and transferred into HPLC vial and was injected onto the LC–MS=MS system for analysis. Chromatographic Conditions Chromatography was performed on a Kromasil-100 C18 analytical column (50 mm Â 4.6 mm i.d., 5 m, Eka Chemicals, Bohus, Sweden). The column was maintained at 30 C. The compounds were eluted isocratically at a flow rate of 0.45 mL=min. The mobile phase consisted of 0.1% v=v ammonia solution:acetonitrile:methanol (10:81:09, v=v=v). Mass Spectrometric Conditions The mass spectrometer was operated in the negative ion detection mode. Nitrogen was used as the nebulizing, turbo spray, and curtain gas, with the optimum values set at 25, 30, and 25 psi, respectively. The tempera- ture of the vaporizer was set at 400 C and ESI needle voltage was adjusted to À4500 V. The declustering potential was set at À80, À85, and À80 V for DHA, EPA, and IS, respectively. Identification was performed using mul- tiple reaction monitoring (MRM) of the transitions of m=z 327.30 ! m=z 283.20 for DHA, m=z 301.30 ! m=z 257.20 for EPA and m=z 265.00 ! m=z 182.20 for nevirapine (IS), respectively, with a dwell time of 200 ms per transition. For collision-induced dissociation (CID), nitrogen was used as
1816 C. Ghosh et al. the collision gas at a pressure of 8 psi. The collision energy was À20 V, À22 V, and À35 V for DHA, EPA, and IS, respectively. Method Validation EPA and DHA, are the endogenous compounds, so the back-calculated concentration and accuracy were calculated by using a validated Microsoft Excel sheet, which was validated by SAS software. Four zero standard sam- ples were injected with each calibration curve to calculate the average area ratio of the endogenous components. The calculated average area ratio was subtracted from the area ratio of each sample to get the actual area ratio of the components, which were further used to back calculate the concen- tration and accuracy of calibration standards and quality control samplesDownloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 using the validated Excel. The sample concentrations were calculated using weighted (1=x2) least squares linear regression. To evaluate linearity, plasma calibration curves were prepared and were assayed on three separate days. Accuracy and precision were also assessed by determining QC samples at three concen- tration levels on three different validation days. The accuracy was expressed by, (observed concentration=spiked concentration) Â 100% and the pre- cision by relative standard deviation (RSD). The extraction recoveries of DHA and EPA at three QC levels were evaluated by comparing peak areas of analytes obtained from plasma samples with the analytes spiked before extraction to those spiked after the extraction. The matrix effect was car- ried out by extracting low and high quality control samples in triplicate from six different plasma sources. The hemolysis effect and lipemic effect experiments were carried out at two different concentration levels. The stability of DHA and EPA in the diluents was assessed by placing QC samples under ambient conditions for at least 6 hr. The freeze–thaw stability of DHA and EPA was also assessed by analyzing QC samples undergoing four freeze (À20 C) and thaw (room temperature) cycles. Similarly stability of DHA and EPA was assessed inside the auto sampler and in plasma at room temperature. Subsequently, the DHA and EPA concentrations were measured and compared with freshly prepared samples, respectively. Clinical Protocol The pharmacokinetic study protocol presented in this paper was approved by the Independent Medical Ethics Committee of Cadila Contract 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 200 mL of drinking water after an overnight fasting of at
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1817 least 10 hr in each period. The test capsule contains 30 mg DHA and 250 mg EPA, whereas the reference capsule contains 250 mg DHA and 350 mg EPA. The administered subject was a healthy, adult, male, human volunteer of Indian origin. In each period, a total of 17 blood samples were collected including four pre-dose sample prior to drug administration. The blood samples were immediately centrifuged at 2000 rpm for 10 min at 4 C, and the plasma samples were stored at À30 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=Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 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 types of columns and mobile phases were used. Length of the column varied from 50 mm to 150 mm, and the particle size varied from 3.5 m to 5 m. Columns of different types of stationary phase such as C8 and C18 were used which showed some remarkable effect on peak shape. Finally, Kromasil-100 C18 analytical column (50 mm Â 4.6 mm i.d., 5 m, particle size) was selected for analysis. The influence of buffer molarities, pH, and types of organic modifier on the signal intensities was also studied. Initially, 90% acetonitrile: 10% of 0.1% ammonia solution (v=v) at a flow rate of 0.500 mL=min was tried but it led to improper resolution between the other endogenous peaks present in the extracted plasma samples. Therefore, methanol was introduced in a small quantity, 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 and methanol (10:81:09, v=v) as the mobile phase at a flow rate of 0.450 mL=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 interfere with the main peak. Then, acetonitrile was used as the precipitating agent which produced a better result. Initially, a conventional 2:1(v=v) ratio of acetonitrile to plasma was used, but due to high extraction recovery, the responses 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,
1818 C. Ghosh et al. according to the U.S. Food and Drug Administration (FDA) guidance for the validation of Bioanalytical methods. Linearity Linearity of calibration standards was assessed by subjecting the spiked concentrations and the respective peak areas using 1=X2 linear least-squares regression analysis. Linearity ranged from 50.00 to 7498.50 ng=mL for DHA and EPA both (r 0.990). In aqueous solution, accuracy of all calibration standards was within 85–115%, except LLOQ where it was 80–120%. Specificity and Selectivity As DHA and EPA are endogenous components, the specificity experimentDownloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 was performed for IS only. Six different lots of normal plasma and one lot of lipemic plasma and hemolyzed plasma were analyzed to ensure that no endogenous interference took place with the mass transitions of the IS. LLOQ level samples along with plasma blank from the respective plasma lot were pre- pared and analyzed. In each plasma blank, the response at the retention time of IS was 5% of the IS response in the respective LLOQ. Figure 2A and 2B represents the plasma blank of DHA and EPA, respectively, with IS. Accuracy and Precision For 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 accuracy of each sample preparation was determined by injection of calibration sam- ples and three QC samples in six replicates for 3 d. Obtained accuracy and precision (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 is within the acceptable limits of 100 Æ 20% of the theoretical value at LLOQ and 100 Æ 15% at all other concentration levels. The precision around the mean value was never greater than 15% at any of the concentration studied. Limit of Quantitation Process and inject six LLOQ and six ULOQ samples along with cali- bration standards in the same range used for calculation of precision and accuracy. 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 was 101.32. For EPA the %CV at LLOQ level was 4.98 and at ULOQ level was 3.74. The average %accuracy at LLOQ level was100.34 and at ULOQ level was 99.30.
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1819Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 FIGURE 2 A) Representative chromatogram of plasma blank of DHA and IS and B) Representative chromatogram of plasma blank of EPA and IS. (Color figure available online.) Recovery Study A 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%,
1820 C. Ghosh et al. 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 5.25 MQC 108.10 2.05 104.61 4.02 HQC 94.35 4.21 96.84 5.03 Day 2 LQC 91.45 2.36 94.81 5.79 MQC 99.69 3.68 99.52 2.50 HQC 97.27 3.95 97.94 4.47 Day 3 LQC 97.71 5.77 100.05 0.92 MQC 104.74 0.93 108.33 1.00 HQC 97.07 2.41 99.35 4.02Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 medium quality control (MQC) level was 47.96%, and for high quality control (HQC) level was 49.60%. The mean recovery of DHA was 52.07%. % coefficients of variation (%CV) of mean recovery of all three QCs 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%. %CV of mean recovery of all three QCs was 5.82. Recovery of internal standard was 42.02%. Matrix Effects Matrix 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 of all LQC samples was 3.14 and the average % accuracy for all HQC level was 100.56 and %CV of all HQC samples was 2.06. Whereas, for EPA, the average % 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 Effects To determine hemolysis effects, six QC samples were prepared in hemo- lyzed plasma with three concentration levels of low, medium, and high. Six replicates of each QC samples were analyzed together with a set of cali- bration standard prepared in normal plasma. The accuracy of each sample preparation was determined by injection of calibration samples and three QC samples in six replicate. For DHA the average % accuracy for LQC level was 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.
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1821 TABLE 2 Summary of Stability Data of DHA Mean Precision Mean Percent Stability Stability QC Level (%CV) Accuracy Change Duration Bench top LQC 4.85 85.72 À9.18 8 Hr HQC 4.09 102.88 5.98 Freeze thaw LQC 5.76 92.70 1.04 4 Cycles HQC 2.01 90.09 2.98 Auto sampler LQC 7.28 97.50 À0.57 24 Hr HQC 1.86 97.27 À0.47 For EPA the average % accuracy for LQC level was 105.31, for MQC level was 111.02, and for HQC level was 103.91. The %CV of LQC was 4.85, for MQC was 1.50, and for HQC was 2.02.Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 Lipemic Effects To 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 of calibration standard prepared in normal plasma. The accuracy of each sample preparation was determined by injection of calibration samples and three QC samples in six replicate. For DHA the average % accuracy for LQC level was 98.73, for MQC level was 106.72, and for HQC level was 105.67. The %CV of LQC was 9.03, for MQC was 2.49, and for HQC was 1.97. For EPA the average % accuracy for LQC level was 93.17, for MQC level was 107.34, and for HQC level was 103.80. The %CV of LQC was 3.11, for MQC was 2.65, and for HQC was 2.53. Stability Studies The 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 3 Summary of Stability Data of EPA Mean Precision Mean Percent Stability Stability QC Level (%CV) Accuracy Change Duration Bench top LQC 1.42 100.36 0.32 8 Hr HQC 3.54 106.41 7.11 Freeze thaw LQC 2.98 98.30 2.36 4 Cycles HQC 1.57 91.33 3.26 Auto sampler LQC 4.79 110.39 1.47 24 Hr HQC 2.31 95.74 À0.82
1822 C. Ghosh et al. freshly processed calibration standards and QC samples. Analytes were considered stable when the change of concentration is Æ10% with respect to their initial concentration. The %CV of DHA at LQC and HQC levels for, bench top stability, auto sampler stability, and freeze-thaw stability were 4.85, 7.82, and 5.76 andDownloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 FIGURE 3 (A) Representative chromatogram of real sample of DHA and (B) Representative chroma- togram of real sample of EPA. (Color figure available online.)
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1823 4.09, 1.86, and 2.01, respectively, whereas, the %CV of PHA at LQC and HQC 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 and EPA, respectively. Calibration Curve Parameters The 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: À0.0984 to 0.2982), respectively. The mean corre- lation coefficient, r was 0.9985 (Range: 0.9965 to 0.9999).Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 FIGURE 4 (A) Plasma concentration-time curves for DHA (n ¼ 1) and (B) Plasma concentration-time curves for EPA (n ¼ 1). (Color figure available online.)
1824 C. Ghosh et al. For EPA the mean slope and y-intercepts were 0.000618 (Range: 0.0005 to 0.0008) and 0.0143 (Range: À0.0629 to 0.0743), respectively. The mean correlation coefficient, r was 0.9984 (Range: 0.9962 to 0.9999). APPLICATION The validated method was applied to determine the concentration time profile, following single dose oral administration of capsule in healthy human volunteer. This developed method can also be applied for estimation of EPA and DHA from urine, serum, and other biological matrices to some extent. This method will be a valuable tool during in vitro study of EPA and DHA. The method can also be helpful for routine analysis of EPA and DHA in quality control departments. The chromato-Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012 grams obtained from the analysis of real samples are presented in Figure 3A and 3B for DHA and EPA, respectively. After LC-MS=MS analy- sis, the plasma concentration of DHA and EPA were measured. Figure 4A and 4B shows the plasma concentration-time curves for DHA and EPA, respectively. CONCLUSION A LC=MS=MS method with simple protein precipitation has been developed for the simultaneous quantitative determination of DHA and EPA in human plasma. The present method affords the rapid, sensitivity, accuracy, and precision necessary for fast quantitative measurements in pharmacokinetic studies and therapeutic monitoring of DHA and EPA. REFERENCES 1. Simopoulos, A. P. Omega-3 Fatty Acids in Health and Disease and in Growth and Development. Am. J. Clin. Nutr. 1991, 54, 438–463. 2. Drevon, C. A. Marine Oils and Their Effects. Nutr. Rev. 1992, 50, 38–45. 3. Chauke, E.; Chkrowska, E.; Thaela-Chimuka, M. J.; Chimuka, L.; Nsengimana, H.; Tutu, H. Fatty Acids Composition in South African Freshwater Fish as Indicators of Food Quality. Water SA. 2008, 34, 119–126. 4. Kurowska, E. M.; Dresser, G. K.; Deutsch, L.; Vachon, D.; Khalil, W. Bioavailability of Omega-3 Essen- tial Fatty Acids from Perilla Seed Oil. Prostaglandins Leukot Essent Fatty Acids. 2003, 68, 207–212. 5. Rusca, A.; Stefano, A. F. D. D.; Doig, M. V.; Scars, C.; Perucca, E. Relative Bioavailability and Phar- macokinetics of Two Oral Formulations of Docosahexaenoic Acid=Eicosapentaenoic Acid After Multiple-Dose Administration in Healthy Volunteers. Eur. J. Clin. Pharmacol. 2009, 65, 503–510. 6. Hidetaka, Y.; Azuma, Y.; Maeda, N.; Kawasaki, H. High-Performance Liquid Chromatographic Determination of Eicosapentanoic Acid in Serum by a Chemiluminescence Labeling Method. Chem. Pharm. Bull. 1998, 36, 1905–1908. 7. Ghosh, C.; Jha, V.; Ahir, R.; Shah, S.; Shinde, C. P.; Chakraborty, B. A Rapid and Most Sensitive Liquid Chromatography=Tandem Mass Spectrometry Method for Simultaneous Determination of
Simultaneous Determination of DHA and EPA by ESI-LC-MS=MS 1825 Alverine and Its Major Metabolite, Para Hydroxy Alverine, in Human Plasma: Application to a Pharmacokinetic and Bioequivalence Study. Drug Test. Anal. 2010, 2, 284–291. 8. Ghosh, C.; Singh, R. P.; Inamdar, S.; Mote, M.; Chakraborty, B. Sensitive, Selective, Precise and Accu- rate LC–MS Method for Determination of Clonidine in Human Plasma. Chromatographia 2009, 69, 1227–1232. 9. Ghosh, C.; Shinde, C. P.; Chakraborty, B. S. Ionization Polarity as a Cause of Matrix Effects, its Removal and Estimation in ESI-LC-MS=MS Bio-analysis. J. Anal. Bioanal. Tech. 2010, 1, 106. 10. Ghosh, C.; Gaur, S.; Shinde, C. P.; Chakraborty, B. A Systematic Approach to Overcome the Matrix Effect During LC-ESI-MS=MS Analysis by Different Sample Extraction Techniques. J. Bioequiv. Availab. 2011, 3, 122–127. 11. Guidance for Industry, Bioanalytical Method Validation (2001). Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Dholka, India 2001.Downloaded by [Chinmoy Ghosh] at 00:20 09 August 2012