Poster Sorrento
1
COMPARISON OF DIFFERENT SORBENTS FOR THE SIMULTANEOUS SPE OF EIGHT NSAIDs FROM HUMAN PLASMA COUPLED WITH HPLC. G. Carlucci , M. D’Ercole, L. Di Federico, P.Iuliani, M. Locatelli Dipartimento di Scienze del Farmaco-Facoltà di Farmacia-Università degli Studi “G. D’Annunzio” di Chieti-Pescara-Via dei Vestini 66100 Chieti-Italy. Introduction Sample preparation remains a central feature of pharmaceutical and biomedical analysis, and the time consuming nature of the current methodology has resulted in a continuous search for new and improved techniques. Solid phase extraction (SPE) is an important area with the introduction of new phases or formats and development of fully automated methods. The bulks of the commercially available phases based on bonded silicas (C18, C8, C4, etc.) have been very successful, whilst the presence of residual silanols often complicates the extraction of ionic compounds because of ionic interactions e.g. 1,3 . Recently a number of non-silica based polymeric SPE sorbents have been introduced which offer a potential for a different range of selectivities to silica based materials. In the present study we developed a SPE-HPLC procedure for the simultaneous analysis of seven widely used antiinflammatory drugs (NSAIDs), namely: furprofen, indoprofen, ketoprofen, fenbufen, flurbiprofen, indomethacin and ibuprofen (Fig. 1). To achieve the most efficient preconcentration of the analytes from human plasma we investigated the retention ability of five commercially available SPE cartridges based on different kinds of sorbents, such as octadecyl-bonded silica (Sep-Pak, Waters), polymeric (Evolute, Argonaut and Bond Elute Plexa, Varian), divinylbenzene-N-vinylpyrrolidone copolimers (Oasis HLB, Waters) and styrene- divinylbenzene polymer (StrataX, Phenomenex). The identification and quantification of the investigated NSAIDs was carried out by means of HPLC coupled with diode array detection (DAD) at isocratic conditions. 1.Ruane, R.J.; Wilson, I.D. J. Pharm. Biomed. Anal. 1987, 5, 723-727. 2. Roberts, D.W.; Ruane, R.J; Wilson, I.D. J. Pharm. Biomed. Anal. 1989, 7, 1077-1086. 3. Martin, P.; Morgan, E.D.; Wilson, I.D. J. Pharm. Biomed. Anal. 1996, 14, 419-427. OH O O ketoprofen OH O furprofen O O N H 3 CO OH O O Cl indometacin indoprofen N O O OH OH O ibuprofen OH O flurbiprofen F O fenbufen OH O Fig. 1 Experimental Separation was performed on a column Hyperclone (250 x 4,6 mm, 5 μm, Phenomenex) at a flow rate of 1 mL min -1 at room temperature, in isocratic conditions using a mixture (50/50 v/v) of acetonitrile and phosphate buffer (10 mM, pH 2,5) as mobile phase. UV spectra were recorded from 190 to 400 nm. The working wavelenght for quantitative analysis of each analyte (Table 1) was selected to have the maximum absorbance and the best peak resolution. Fig. 2 shows the HPLC-DAD chromatogram of a standard solution of the seven analytes extracted at appropriate wavelenght (@218 nm). Calibration curves of all analytes were obtained from triplicate experiments using ten standard samples in the concentration range 25-250 μg mL -1 obtained by appropriate dilution of working standard solutions with mobile phase. The relevant chromatographic parameters were obtained by averaging the results of three chromatographic runs. The SPE sorbents were conditioned with 1 mL of methanol and equilibrated with 1 mL of phosphate buffer (pH 7,4; 10 mM). The solvents were forced through the cartridge at a rate of 1 mL min -1 . The spiked plasma sample (1 mL) was added with 2 mL of acetonitrile and two drops of phosphoric acid for protein precipitation and it was centrifugated at twice at 2100 x g. The surnatant was concentrated to the volume of 1 mL and passed trough the SPE cartridge approximately at a flow rate of 0,5 mL min -1 . The cartridge was then washed with a mixture of methanol/phosphate buffer 10 mM pH 7,4 (5:95 v/v) and dried for a few minutes. The retained analytes were eluted by 1 mL of methanol. The eluates were evaporated to dryness at room temperature under vacuum. The dry residue was finally dissolved in 100 µL of HPLC mobile phase. 20 L aliquots were injected into chromatograph for evaluation of the extraction recovery. Results and discussion For the development of SPE method and for the comparison of the different sorbents three concentrations levels were studied, taking into account of the normal plasmatic levels of NSAIDs in treated patients. The average extraction recoveries obtained from experiments carried out in triplicate are collected in Table 2. Apparently the overall performance of the selected cartridges is not appreciably influenced by drug concentration. The best recoveries were provided by Oasis HLB, Bond-Elut and Strata X. Sorbent (spiked level (μg m L -1 )) O asis 2,5 10 17,5 Evolute 2,5 10 17,5 Strata X 2,5 10 17,5 Bond-elut 2,5 10 17,5 Sep-Pak 2,5 10 17,5 furprofen 38 43 37 40 38 35 70 65 68 66 69 66 23 21 22 indoprofen 92 97 88 13 20 17 78 81 73 92 101 89 25 28 22 ketoprofen 108 99 101 45 46 51 99 105 98 102 95 92 69 77 62 fenbufen 93 90 89 36 32 29 85 82 89 99 93 98 79 75 80 flurbiprofen 83 78 90 43 47 38 68 75 73 80 82 86 60 63 57 indom ethacin 70 75 77 54 55 60 81 66 76 99 94 97 69 72 75 ibuprofen 88 85 92 37 41 42 72 70 84 85 89 83 66 61 59 Table 2 Comparison of retention ability of the five selected sorbents furprofen indoprofen ketoprofen fenbufen flurbiprofe n indomethacin ibuprofen Fig. 2 Chromatogram of a standard solution of NSAIDs extracted @ 218 nm. Table 1 Analytical data for the HPLC method N SAID s t R (m in) λ (nm ) r 2 LO D (μg/m L) LO Q (μg/m L) furprofen 4.1 291 0.9991 0.5 1.5 indoprofen 4.6 280 0.9987 0.05 0.5 ketoprofen 6.4 256 0.9994 1.5 2.5 fenbufen 7.6 283 0.9967 1.0 1.5 flurbiprofen 10.4 246 0.9995 2.5 2.5 indom etacina 12.1 318 0.9995 2 2.5 ibuprofen 14.1 219 0.9991 2.5 2.5
Transcript of Poster Sorrento
COMPARISON OF DIFFERENT SORBENTS FOR THE SIMULTANEOUS SPE OF EIGHT NSAIDs FROM HUMAN PLASMA
COUPLED WITH HPLC.G. Carlucci, M. D’Ercole, L. Di Federico, P.Iuliani, M. Locatelli
Dipartimento di Scienze del Farmaco-Facoltà di Farmacia-Università degli Studi “G. D’Annunzio” di Chieti-Pescara-Via dei Vestini 66100
Chieti-Italy.
IntroductionSample preparation remains a central feature of pharmaceutical and biomedical analysis, and the time consuming nature of the current methodology has resulted in a continuous search for new and improved techniques. Solid phase extraction (SPE) is an important area with the introduction of new phases or formats and development of fully automated methods. The bulks of the commercially available phases based on bonded silicas (C18, C8, C4, etc.) have been very successful, whilst the presence of residual silanols often complicates the extraction of ionic compounds because of ionic interactions e.g.1,3. Recently a number of non-silica based polymeric SPE sorbents have been introduced which offer a potential for a different range of selectivities to silica based materials. In the present study we developed a SPE-HPLC procedure for the simultaneous analysis of seven widely used antiinflammatory drugs (NSAIDs), namely: furprofen, indoprofen, ketoprofen, fenbufen, flurbiprofen, indomethacin and ibuprofen (Fig. 1). To achieve the most efficient preconcentration of the analytes from human plasma we investigated the retention ability of five commercially available SPE cartridges based on different kinds of sorbents, such as octadecyl-bonded silica (Sep-Pak, Waters), polymeric (Evolute, Argonaut and Bond Elute Plexa, Varian), divinylbenzene-N-vinylpyrrolidone copolimers (Oasis HLB, Waters) and styrene-divinylbenzene polymer (StrataX, Phenomenex). The identification and quantification of the investigated NSAIDs was carried out by means of HPLC coupled with diode array detection (DAD) at isocratic conditions.
1.Ruane, R.J.; Wilson, I.D. J. Pharm. Biomed. Anal. 1987, 5, 723-727.
2. Roberts, D.W.; Ruane, R.J; Wilson, I.D. J. Pharm. Biomed. Anal. 1989, 7, 1077-1086.
3. Martin, P.; Morgan, E.D.; Wilson, I.D. J. Pharm. Biomed. Anal. 1996, 14, 419-427.
OH
O
O
ketoprofen
OH
O
furprofenO
O N
H3CO OH
O
O
Cl
indometacin
indoprofen
N
O
O
OH
OH
O
ibuprofen
OH
O
flurbiprofen
F
O
fenbufen
OH
O
Fig. 1
Experimental Separation was performed on a column Hyperclone (250 x 4,6 mm, 5 μm, Phenomenex) at a flow rate of 1 mL min -1 at room temperature, in isocratic conditions using a mixture (50/50 v/v) of acetonitrile and phosphate buffer (10 mM, pH 2,5) as mobile phase. UV spectra were recorded from 190 to 400 nm. The working wavelenght for quantitative analysis of each analyte (Table 1) was selected to have the maximum absorbance and the best peak resolution. Fig. 2 shows the HPLC-DAD chromatogram of a standard solution of the seven analytes extracted at appropriate wavelenght (@218 nm). Calibration curves of all analytes were obtained from triplicate experiments using ten standard samples in the concentration range 25-250 μg mL -1 obtained by appropriate dilution of working standard solutions with mobile phase. The relevant chromatographic parameters were obtained by averaging the results of three chromatographic runs.
The SPE sorbents were conditioned with 1 mL of methanol and equilibrated with 1 mL of phosphate buffer (pH 7,4; 10 mM). The solvents were forced through the cartridge at a rate of 1 mL min-1. The spiked plasma sample (1 mL) was added with 2 mL of acetonitrile and two drops of phosphoric acid for protein precipitation and it was centrifugated at twice at 2100 x g. The surnatant was concentrated to the volume of 1 mL and passed trough the SPE cartridge approximately at a flow rate of 0,5 mL min -1. The cartridge was then washed with a mixture of methanol/phosphate buffer 10 mM pH 7,4 (5:95 v/v) and dried for a few minutes. The retained analytes were eluted by 1 mL of methanol. The eluates were evaporated to dryness at room temperature under vacuum. The dry residue was finally dissolved in 100 µL of HPLC mobile phase. 20 L aliquots were injected into chromatograph for evaluation of the extraction recovery.Results and discussionFor the development of SPE method and for the comparison of the different sorbents three concentrations levels were studied, taking into account of the normal plasmatic levels of NSAIDs in treated patients. The average extraction recoveries obtained from experiments carried out in triplicate are collected in Table 2. Apparently the overall performance of the selected cartridges is not appreciably influenced by drug concentration. The best recoveries were provided by Oasis HLB, Bond-Elut and Strata X.
Sorbent (spiked level (μg mL-1))
Oasis 2,5 10 17,5
Evolute 2,5 10 17,5
Strata X 2,5 10 17,5
Bond-elut 2,5 10 17,5
Sep-Pak 2,5 10 17,5
furprofen 38 43 37 40 38 35 70 65 68 66 69 66 23 21 22
indoprofen 92 97 88 13 20 17 78 81 73 92 101 89 25 28 22
ketoprofen 108 99 101 45 46 51 99 105 98 102 95 92 69 77 62
fenbufen 93 90 89 36 32 29 85 82 89 99 93 98 79 75 80
flurbiprofen 83 78 90 43 47 38 68 75 73 80 82 86 60 63 57
indomethacin 70 75 77 54 55 60 81 66 76 99 94 97 69 72 75
ibuprofen 88 85 92 37 41 42 72 70 84 85 89 83 66 61 59
Table 2 Comparison of retention ability of the five selected sorbents
furp
rofe
n
ind
op
rofe
n
keto
pro
fen
fen
bu
fen
flu
rbip
rofe
n
ind
om
eth
acin
ibu
pro
fen
Fig. 2 Chromatogram of a standard solution of NSAIDs extracted @ 218 nm.
Table 1 Analytical data for the HPLC method
NSAIDs tR (min) λ (nm) r2 LOD (μg/mL) LOQ (μg/mL)
furprofen 4.1 291 0.9991 0.5 1.5
indoprofen 4.6 280 0.9987 0.05 0.5
ketoprofen 6.4 256 0.9994 1.5 2.5
fenbufen 7.6 283 0.9967 1.0 1.5
flurbiprofen 10.4 246 0.9995 2.5 2.5
indometacina 12.1 318 0.9995 2 2.5
ibuprofen 14.1 219 0.9991 2.5 2.5
