Method for Determination of Aspirin and Salicylic Acid in Rat Whole Blood by High Pressure Liquid Chromatography

This article was downloaded by: [171.67.34.69] On: 06 May 2013, At: 16:31 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Analytical Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lanl20 Method for Determination of Aspirin and Salicylic Acid in Rat Whole Blood by High Pressure Liquid Chromatography Chau-Hwei J. Fu a , Srikumaran Melethil a & William D. Mason a a Pharmacokinetics Laboratory, University of Missouri-Kansas City Schools of Pharmacy & Medicine, 2411 Holmes, KC, MO, 64108 Published online: 05 Dec 2006. To cite this article: Chau-Hwei J. Fu , Srikumaran Melethil & William D. Mason (1985): Method for Determination of Aspirin and Salicylic Acid in Rat Whole Blood by High Pressure Liquid Chromatography, Analytical Letters, 18:3, 269-277 To link to this article: http://dx.doi.org/10.1080/00032718508066130 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial 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 representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. ANALYTICAL LETTERS, 18(B3), 269-277 (1985) METHOO FOR DETERMINATION OF ASPIRIN AND SALICYLIC ACID I N RAT WHOLE BLOOD BY HIGH PRESSURE LIQUID CHROMATOGRAPHY KEY WORDS: Asp i r in , S a l i c y l i c acid, HPLC, Rat Whole Blood bY Chau-Hwei J. Fu Srikumaran Me le th i l Wi l l iam 0. Mason Pharmacokinetics Laboratory Un ivers i ty o f Missour i-Kansas C i t y Schools o f Pharmacy & Medicine 2411 Holmes, KC, MO 64108 ABSTRACT A method has been developed t o s imultaneously determine a s p i r i n and s a l i c y l i c a c i d i n r a t whole blood. A s p i r i n and s a l i c y l i c a c i d a r e e x t r a c t e d f r o m a c i d i f i e d w h o l e b l o o d i n t o a 50150 v / v e t h y 1 acetatet ’buty I c h 1 o r i d e organ ic .so1 vent system c o n t a i n i n g i n t e r n a l s tandard (me ta -an is i c ac id ) . F o l l o w i n g c o n t r o l l e d e v a p o r a t i o n o f t h e organic ex t rac t under p a r t i a l vacuum, the d r ied residue i s recons t i tu ted w i th mob i le phase. Chromatography i s i on suppression reverse phase on a 5 pm octy ldecas i land column wi th de tec t ion by UV absorbance a t 280 nm. I n t h i s method, t h e procedure o f b l o o d c e n t r i f u g a t i o n f o r p lasma p r e p a r a t i o n i s e l im ina ted . Therefore, t h e b l o o d volume r e q u i r e d i s decreased and t h e s e n s i t i v i t y o f a n a l y s i s i s c o n s i d e r a b l y increased. Concent ra t ions o f a s p i r i n as low as 0.5 mcg/ml and s a l i c y c l i c a c i d as l ow as 1.5 rncglml can be quant i ta ted i n as l i t t l e as 100p&o f whole blood. 269 Copyright 0 1985 by Mprcel Dekka, Inc. 0003-27 19/8S/l803-0269$3.50/0 D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 270 FU, MELETHIL, AND MASON Aspirin is one of the m s t extensively used drugs. A method of high pressure liquid chromatography (HPLC) for the analysis of aspirin and its metabolites in human plasma and urine was developed in our laboratory in the late 1970s and revised in the early 1980s improve the sensitivity and reproducibility, this method can be applied to measure aspirin and salicylic acid in rat whole blood. studies of kinetics and metabolism of aspirin in the rat are both possible and the extrapolation of these results to humans can be meaningful. . With modifications to 1,2 Therefore, the The HPLC method described herein has been employed to measure aspirin (ASA) and salicylic acid (SA) in more than 500 rat whole blood samples collected during a pharmacokinetic study. EXPERIMENTAL Materials-Aspirin was obtained from Miles Laboratories (El khart, Indi- ana). Salicylic acid (A.C.S.) and amnonium sulfate (A.C.S.) were obtained from Fisher Scientific Company (Fair Lawn. N.J.). Butyl chloride, ethyl acetate and acetonitrile were obtained from Burdick and Jackson Laboratories (Muskegon, Michigan). Company (Milwaukee, Wisconsin). Glacial acetic acid (100%) and ortho- phosphoric acid (85%) were obtained from J.T. Baker Chemical Company (Phil- lipsburg, N.J.). Sodium fluoride was obtained from Mallinckrodt Inc. (Paris, Kentucky), All chemicals were used as obtained with no further purification. Meta-anisic acid was obtained from Aldrich Chemical Apparatus-A high pressure liquid chromatography system consisting of a model 6000A solvent delivery pump and a 710A automatic injector (Waters Asso- ciates, Milford, Massachusettes) were used with a 15 cm, 5 ym octyldecasilane ultrasphere column (Altex, Berkley, Calif.) to effect chromatographic separ- ation. A model 7 7 3 UV absorbance detector by Schoeffel Instruments (Div. of Kratos, Inc., Ramsey, N.J.) was employed. A model C-R1A recording in- tegrator (Shimadzu corporation, Kyoto, Japan) was used to record and integrate peak areas. D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 DETERMINATION OF ASPIRIN AND SALICYLIC ACID 271 Specimen Collection and Preparation-Since aspirin is rapidly hydrolyzed in blood and plasma to salicylic acid, one very important factor is the re- quirement that specimens be collected, handled and stored under conditions which minimize aspirin hydrolysis. collected in chilled 15 ml conical centrifuge tubes, along with 0.1 ml of 50/50 v/v phosphoric acid containing 0.25 g/ml of ammonium sulfate and 2.5 mg/ml of sodium fluoride. Specimens were immediately mixed by vortexing (30 seconds) and frozen at -30°C until analysis. be completed within 1 minute of collection to prevent hydrolysis. Precise quantities of blood (100 PE) were Specimen preparation should When processing large numbers of samples, calibration standards should be prepared within 24 hours of sample collection, stored with unknown samples and all assays completed within two weeks. In this manner, daily calibration of the method compensates for the aspirin hydrolysis. If multiple determina- tions are desired, samples should be split prior to freezing to allow only one thaw per analysis. Whole Blood Extraction Procedure-This procedure has been optimized to measure aspirin in the 0.5 to 40.0 mcg/ml and salicylic acid in the 1.5 to 120.0 mcg/ml range, respectively. in butylchloride extraction solvent containing 0.2 mcg/ml meta-anisic acid as internal standard was added to each tube. texing two minutes and centrifugation for 10 minutes. Exactly 6.0 ml of a 50/50 v/v ethyl acetate Extraction was effected by vor- The organic (top) layer was transferred to a second 15 ml concical centrifuge tube and evaporated to dryness under vacuum in a vortex evap- orator at 35OC temperature and 29.mmHg with the time being dependent on the number of tubes. from 60 minutes for 7 tubes to 90 minutes for 36 tubes. ic acid and meta-anisic acid can be lost to sublimation during this step if excessive time, temperature or vacuum are employed. of the dry-down must be tailored to the particular apparatus and number of Generally speaking, the range of time required are Aspirin, salicyl- Actual conditions D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 272 FU, MELETHIL, AND MASON samples. phase and could be s to red (4'C) fo r 24 hours p r i o r t o chromatography with- out l oss o f asp i r in . using the automatic i n jec to r . Af ter dry-down, the residue was d isso lved i n 0.6 m l of mobi le F i n a l l y . 100 V E of t h i s so lu t i on was i n j e c t e d by Chromatography-A mobi le phase composed of 10% a c e t o n i t r i l e and 5% g l a c i a l a c e t i c a c i d a t a f l ow r a t e o f 0.7 ml/min provides 625 and 595 theo re t i ca l p la tes f o r a s p i r i n and s a l i c y l i c ac id per cm o f column on the 5 wn oc ta ldecy ls i lane . With the capac i ty fac to rs of 6.51 f o r a s p i r i n and 8.39 f o r s a l i c y l i c ac id on the 15 cm column, good r e s o l u t i o n from co-extracted compounds has been observed. between i n j e c t i o n s as samples from some animals show l a t e e l u t i n g peaks which may i n t e r f e r e i n subsequent chromatograms. i n t e r v a l was selected i n many r a t s t o avo id an unknown peak e lu ted a t around 64 minutes a f t e r i n jec t i on . The t y p i c a l pre-dose and 2.5 minute post-dose chromatograms are shown i n f i g u r e 1. Care must be taken i n se lec t i ng the i n t e r v a l For example, a 48 minute De tec t i on -U l t rav io le t absorbance a t 280 nm g ives the acceptable response f o r asp i r i n , s a l i c y l i c a c i d and meta-anisic acid. Calculat ion, L i n e a r i t y and Reproducibility-Quantitation was made by computing the r a t i o s o f a s p i r i n and s a l i c y l i c a c i d peak areas to meta-anis ic ac id peak area and comparing w i t h those of c a l i b r a t i o n standards. Ca l ib ra- t i o n standards and blank whole blood samples were analyzed w i t h each sample run. t o added drug; the response being reproduc ib le from day t o day. of the unknowns was achieved us ing l i n e a r regression ana lys i s f o r t he l i n e y = ax + b, where y equals the concentrat ion, x equals the r a t i o o f peak area of t he component t o the peak area o f t he i n t e r n a l standard, a equals the slope, and b i s the in te rcept . Table I and I 1 present the r a t i o s and regression s t a t i s t i c s f o r a recen t l y conducted study i n which a s p i r i n was administered t o ra ts . Both r a t i o s were l i n e a r (40 mcg/ml ASA and 120 mcg/ml SA) with respect Ca lcu la t i on D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 DETERMINATION OF ASPIRIN AND s A L I c y L I c ACID 273 A. r 6. r T m-AA ASA SA m-AP. FIG. 1 Typical chromatograms for rat whole blood extracts A. Pre-dose sample; 6. Sample computed to contain 2.50 mcg/ml aspirin and 20.50 mcg/ml salicylic acid. Specificity-Aspirin, salicylic acid and meta-anisic acid were well resolved from each other. Using rat whole blood specimens from more than 30 animals participating in a variety of studies, only three pre-dQse samples have shown a peak which interferes with aspirin; however, an interfering peak equivalent to an average 0.52 mcg/ml SA was present in many samples. D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 N U c TA BL E I RE GR ES SI ON S TA TI ST IC S OF C AL IB RA TI ON C UR VE S FO R AS PI RI N (A SA ) Co nc en tr at io n Ra ti o (p k. ar ea A SA /p k. ar ea m- AA ) Me an S. D. %C V 0. 0 m cg /m l 0. 00 00 0. 00 00 0. 00 00 0. 00 00 0. 5 0. 03 52 0. 02 85 0. 02 54 0. 02 96 1. 0 0. 06 55 0. 05 29 0. 06 91 0. 06 96 5. 0 0. 26 45 0. 26 52 0. 26 62 0. 29 16 10 .0 0. 51 16 0. 50 84 0. 51 52 0. 57 57 20 .0 0. 98 88 0. 99 11 1. 01 71 1. 11 09 40 .0 2. 01 45 1. 98 91 2. 15 82 2. 26 68 0. 00 00 0. 02 22 0. 06 10 0. 28 88 0. 55 76 1. 09 77 2. 18 12 ~~ 0. 00 00 0. 02 67 0. 06 30 0. 28 81 0. 51 11 1. 01 05 2. 03 08 0. 00 00 0. 02 79 0. 06 35 0. 27 74 0. 52 99 1. 03 60 2. 10 68 ~~ 0. 00 00 0. 00 0. 00 44 15 .7 7 0. 00 62 9. 76 0. 01 33 4. 79 0. 02 91 5. 49 0. 05 42 5. 23 0. 11 13 5. 28 n N st d. 7 7 7 7 7 7 0. 99 99 0. 99 99 0. 99 95 0. 99 99 0. 99 99 0. 99 98 0. 99 98 0. 00 02 0. 02 Co rr el at io n Co ef fi ci en t S1 op e 20 .0 07 8 20 .1 73 7 18 .6 91 4 17 .7 40 8 18 .3 51 0 19 .8 07 7 19 .1 28 7 1. 00 48 5. 25 In te rc ep t -0 .1 61 7 -0 .1 24 3 0. 11 10 -0 .0 81 3 -0 .1 04 3 -0 .1 92 6 -0 .0 92 2 0. 10 72 11 6. 27 S. E. E. 0. 21 73 0. 14 59 0. 49 60 0. 20 79 0. 14 96 0. 27 13 0. 24 80 0. 13 01 52 .4 6 N st d. = th e nu mb er o f ca li br at io n st an da rd s un de r li ne ar r eg re ss io n S.E .E. = st an da rd e rr or o f es ti ma te E m r I4 Z H 6 0 z D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 TA BL E I1 RE GR ES SI ON S TA TI ST IC S OF C AL IB RA TI O N CU RV ES FO R SA LI CY LI C AC IO ( SA ) C on ce nt ra ti on R at io ( pk .a re a SA /p k. ar ea m- AA ) M ea n S.O . %C V 0. 0 m cg lm l 0. 01 78 0. 02 80 0. 04 35 0. 05 23 0. 06 66 0. 05 41 0. 04 37 0. 01 80 41 .1 9 1.5 0. 14 20 0. 14 64 0. 16 57 0. 14 98 0. 16 26 0. 16 18 0. 15 47 0. 00 99 6. 40 3.0 0. 27 33 0. 25 72 0. 27 19 0. 23 53 0. 25 76 0. 26 78 0. 26 05 0. 01 42 5. 45 15 .0 1. 29 14 1. 28 80 1. 18 89 1. 18 59 1. 21 99 1. 12 42 1. 21 64 0. 06 47 5. 32 30 .0 2. 44 00 2. 47 60 2. 44 22 2. 27 22 2. 42 46 2. 21 81 2. 37 89 0. 10 63 4. 47 60 .0 4. 68 40 4. 64 02 4. 70 67 4. 76 96 4. 69 96 4. 65 12 4. 69 19 0. 04 63 0. 99 12 0. 0 9. 69 20 9. 81 45 9. 65 56 9. 23 51 9. 27 96 9. 24 40 9. 48 68 0. 26 20 2. 76 N st d. 7 7 7 7 7 7 0. 99 98 0. 99 95 0. 99 98 0. 99 98 0, 99 99 0. 99 98 0. 99 98 0. 00 01 0. 01 C or re la ti on C o ef fi ci en t S1 op e 12 .4 83 4 12 .3 51 9 12 .5 16 2 12 .9 78 8 12 .9 90 4 13 .0 17 7 12 .7 23 1 0. 30 39 2. 39 In te rc ep t -0 .2 78 5 -0 .1 23 8 -0 .2 47 4 -0 .4 03 5 -0 .8 23 3 -0 .1 69 9 -0 .3 41 1 0. 25 52 74 .8 2 S.E .E. 0. 95 19 1. 41 56 0. 72 24 0. 84 60 0. 41 37 0. 73 63 0. 84 77 0. 33 16 39 .1 2 * - - - - - ~~ ~~ * 8 E 3 H H cl Po H U N s td . = th e nu m be r o f ca li b ra ti o n s ta nd ar ds u nd er l in e a r re gr es si on S. E. E. = st an da rd e rr o r o f es ti m at e N U cn D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 The r e t e n t i o n times were dependent on the room temperature. the re ten t i on times were: ASA 19.0 minutes; SA 24.0 minutes; and m-AA 31.0 minutes. change i n the shape o f SA peak over time. General ly speaking, Also area i n t e g r a t i o n i s necessary ra the r than peak he igh t due t o SUMMARY Since a s p i r i n i s r a p i d l y hydrolyzed i n blood and plasma, as prev ious ly mentioned, precaut ions must be taken du r ing sample c o l l e c t i o n and ana lys is t o avoid l o s s o f Components. evaporation o f t he organic solvent. parameters o f t ime , temperature and vacuum t o avoid subl imat ion and thus poor assay precis ion. methyl t - b u t y l e ther t o be as e f f i c i e n t an e x t r a c t i o n solvent as bu ty l ch lo r i de - e thy lace ta te mix tu re and i t d r i e s down more q u i c k l y w i t h l ess po ten t i a l f o r subl imat ion problems assay s p e c i f i c i t y i n r a t whole blood are needed. s a l i c y l i c ac id o n l y i s t o be quant i tated, f luorescence de tec t ion may be recom- mended. g ive the most sens i t i ve response and an except iona l l y c lean chromatogram i n The most d e l i c a t e step i n the procedure i s the Great care must be taken t o ad jus t the Pre l im inary evaluat ions i n human plasma assay have shown 2 . Further eva lua t ion o f t h i s so lvent and e f fec ts on For those studies when Fluorescence w i t h e x c i t a t i o n o f 305 nm and 340 nm emission f i l t e r n L human plasma . Unl ike human subjects, the volume of b lood i n r a t s i s q u i t e l i m i t e d which r e s t r i c t s the number of samples one can c o l l e c t w i thout changing the phys io log ica l parameters i n pharmacokinetic studies. The method reported by N iaz i and Bakar employs the procedure o f b lood cen t r i f uga t ion f o r plasma preparat ion, which i s t ime consuming and invo lves the l oss of b lood and o ther components of i n t e r e s t (ASA, SA) i n the sediment. The new method proposed here e l im ina tes t h i s step, therefore the blood consumption i s s i g n i f i c a n t l y decreased and the s e n s i t i v i t y o f ana lys is i s considerably increased. 3 D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13 DETERMINATION OF ASPIRIN AND SALICYLIC ACID REFERENCES 1. Amick, E.N. and Mason, W.D. , Anal. Letters, 12,(B6), 629-640 (1979) 2. Mason, W.D., and Gill i lan R . , Anal. Letters, 16.(812), 903-912 (1983) 3. Bakar, S.K. and Niazi, S . , J. Pharm. Sci . , 72(9), 1020-1023 (1983) Received November 2 7 , 1984 Accepted November 28, 1984 277 D ow nl oa de d by [1 71 .67 .34 .69 ] a t 1 6:3 1 0 6 M ay 20 13