676 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 DRUGS, COSMETICS, FORENSIC SCIENCES Applition of Liqui Chromtogrphy to the Simultneous Determintion of Aetylsliyli Ai, Cffeine, Coeine, Pretmol, Pyrioxine, n Thimine in Phrmeutil Preprtions NATIVIDAD RAMOS-MARTOS,FRANCISCO AGUIRRE-GÓMEZ, n ANTONIO MOLINA-DÍAZ 1 University of Jén, Fulty of Experimentl Sienes, Deprtment of Physil n Anlytil Chemistry, E-23071 Jén, Spin LUIS F. CAPITÁN-VALLVEY University of Grn, Fulty of Sienes, Deprtment of Anlytil Chemistry, E-18071 Grn, Spin This pper esries rpi reverse-phse liqui hromtogrphi metho, with UV etetion, for the simultneous etermintion of etylsliyli i, ffeine, oeine, pretmol, pyrioxine, n thimine in phrmeutil preprtions. A reverse-phse C 18 Nuleosil olumn is use. The moile phse onsists of 2 suessive elunts: wter (5 min) n etonitrile wter (75 + 25, v/v; 9 min), oth juste to ph 2.1 with phosphori i. Before etermintion etylsliyli i is ompletely onverte to sliyli i y lkline hyrolysis. Sliyli i, ffeine, pretmol, pyrioxine, n thimine re ll etete t 285 nm, wheres oeine is etete t 240 nm. Clirtion urves were liner for sliyli i, ffeine, pretmol, n pyrioxine in the rnge of 50 500 mg/l, n for oeine n thimine in the rnge of 50 1000 mg/l. The metho ws pplie to the nlysis of 13 fortifie ommeril phrmeutil preprtions. Reoveries rnge from 92.6 to 105.5%, with reltive stnr evitions of 1.1 5.8%. Comintions of nlgesis s tive priniples in ommeril phrmeutil preprtions usully ontin 2 or more of the most ommon, i.e., etylsliyli i (ASA), sliylmie, pretmol (PCT), n oeine (CO), together with entrl nervous system stimulnts, e.g., ffeine (CF). Gs hromtogrphy (1, 2) n liqui hromtogrphy (LC; 3 5) hve een use for the etermintion of these nlgesis. However, the LC methos evelope for this purpose el usully with only 2 or 3 ompouns (5 7). UV etetion is usully use (8 12), sometimes with preolumn erivtiztion (13). Reeive April 24, 2000. Aepte y JM July 20, 2000. 1 Author to whom orresponene shoul e resse; e-mil: molin@ujen.es. Some vitmins of the B-group, e.g., thimine (TH) n pyrioxine (PY), re foun long with nlgesis or entrl nervous system stimulnts in phrmeutil preprtions. LC provies numerous methos for the seprtion n etermintion of vitmins of the B-group in ifferent mtrixes: foos (14 16), meil foos (17), infnt formul (18), n phrmeutils (19, 20). Nevertheless, only one metho hs een pulishe for the simultneous etermintion of oth wter-solule vitmins n nlgesis or entrl nervous system stimulnts (9). The metho llows the etermintion of only 2 of the ove mentione nlgesis n 1 vitmin B in phrmeutil preprtion. In this pper, we esrie n LC metho for the simultneous etermintion of 6 tive priniples in phrmeutils. Three of them re nlgesis: ASA, CO, n PCT; 2 re wter-solule vitmins: PY n TH; n one is entrl nervous system stimulnt: CF. The metho esrie is sensitive, rpi, n relile n provies urte results in nlyses of phrmeutil preprtions. Experimentl Regents () Stok solutions. Stok solutions t 1.000 g/l for sliyli i (SA; Fluk, Mri, Spin), CF (Pnre, Brelon, Spin), PY hyrohlorie (Fluk), n PCT (Fluk), n t 10.000 g/l for CO (Aelló, Mri, Spin) n TH hyrohlorie (Fluk) were prepre y issolution of the pproprite mounts in wter (LC gre). All hemils were nlytil gre, n ultrpure wter ws use. Stok solutions of vitmins were store t 4 C. Working solutions were prepre ily y suitle ilution. () Moile phses. Methnol (Fluk), etonitrile (Fluk), n ultrpure wter were use. Phosphori i (Pnre, Mri, Spin) ws e to just the ph of the moile phses fter the etonitrile n wter were mixe. Ultrpure wter ws otine from Milli-Q Plus system (Millipore, Mri, Spin; LC gre).
RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 677 Tle 1. LC prmeters otine uner the operting onitions of the evelope metho Anlyte Retention time, t R(min) Cpity ftor, k Asymmetry ftor, SF Resolution, R s Thimine 4.297 0.480 3.2 Pyrioxine 7.085 1.438 2.6 Pretmol 11.251 2.978 1.25 Coeine 11.690 3.315 1.9 Cffeine 12.150 3.301 3 Sliyli i 13.363 3.375 2.64 7.93 19.01 3.50 3.70 10.74 Flow rte of moile phse = 1 ml/min; olumn temperture = 35 C. k =(t R t 0 )/t 0, where t R = retention time of eh ompoun, n t 0 = retention time of the elunt (unretine ompoun). SF = rtio of the 2 hlf-withs t 10% pek height. R s =2 Z/(W +W ), where Z = istne etween the mxim of 2 onseutive peks, n W n W = pek withs. Figure 1. Liqui hromtogrm otine y the evelope metho n showing the seprtion of the 6 ompouns. Conitions: etetor 285 nm; onentrtion of eh ompoun 200 mg/l. Peks: 1=TH;2=PY;3=PCT;4=CO;5= CF; n 6 = SA.
678 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Tle 2. Sttistil prmeters of lirtion urves otine for the evelope metho Anlyte Interept Slope SD SD r F Sliyli i 1.04 4.85 9.08 0.022 0.9998 1.27 Cffeine 59.08 11.84 25.54 0.091 0.9994 6.40 Coeine 89.48 6.15 19.34 0.037 0.9996 1.59 Pretmol 5.29 4.41 9.08 0.032 0.9995 2.37 Pyrioxine 40.13 14.20 11.77 0.041 0.9999 2.23 Thimine 8.37 2.66 4.38 0.008 0.9999 1.68 Stnr evition of the interept. Stnr evition of the slope. Correltion oeffiient. F rtio. Apprtus () Liqui hromtogrph. Moel HP 1050 (Hewlett-Pkr Co., Aronle, PA). () LC olumn. Nuleosil C 18 stinless steel, 250 4.6 mm, 5 µm prtile size (No. 2515; Shrlu Siene, Brelon, Spin). () Asorne etetor. Hewlett-Pkr ioe-rry etetor HP 1040 M series II with vrile wvelength rnge of 200 600 nm. Spetr of the elutes n sorne mesurements t 285 n 240 nm were otine t time intervls of 0.640 s. Tretment of Smples The ontents of psules re quntittively trnsferre, n tlets re rushe to fine power for issolution in wter y sonition. The solutions re then filtere through 0.45 µm pore size Millipore filter, n the filtrtes re ilute to pproprite volume with LC gre wter. To otin the omplete trnsformtion of ASA to SA, lkline hyrolysis is performe for phrmeutils ontining ASA y treting the smple with 1M NOH solution n heting t 60 C for 30 min. Suitle ilutions re me in ll ses efore the smple solutions re injete. LC Conitions The following proeure is use for ll smples: wter (ph 2.1) s the moile phse for 5 min n then etonitrile wter (75 + 25, v/v) for 9 min, t flow rte of 1 ml/min. The etetion wvelength is set t 285 nm for SA, CF, PCT, PY, n TH n t 240 nm for CO. The working olumn temperture is 35 C. The smple volume injete is 10 µl. The hromtogrphi prmeters otine uner these onitions re summrize in Tle 1. Tle 3. Anlytil prmeters lulte for the evelope metho R DL, mg/l Compoun LDR, mg/l Anlyte onentrtion, 100 mg/l Anlyte onentrtion, 400 mg/l Test I Test II e Sliyli i 50 500 1.65 1.47 27 5.1 Cffeine 50 500 5.84 2.84 13 4.9 Pyrioxine 50 500 2.12 1.87 27 10.5 Thimine 50 1000 3.24 4.21 f 19 7.6 Coeine 50 1000 1.11 1.25 f 20 7.7 Pretmol 50 500 3.51 2.33 17 6.6 LDR = liner ynmi rnge. RSD = reltive stnr evition. DL = etetion limit. From ref. 21. e From ref. 22: DL = S y/x n 2 n 1 ; = slope; n S y/x = stnr evition of y-resiuls. f Anlyte onentrtion = 800 mg/l.
RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 679 Tle 4. Reovery of sliyli i, ffeine, pyrioxine, thimine, oeine, n pretmol from syntheti mixtures Sliyli i Cffeine Pyrioxine Thimine Coeine Pretmol Anlyte foun, mg/l Anlyte foun, mg/l Anlyte foun, mg/l Anlyte foun, mg/l Anlyte foun, mg/l Anlyte foun, mg/l Level of eh nlyte e, mg/l 50 50.0 100 ± 2 46.5 93 ± 1 50.3 100.6 ± 0.3 49.3 96 ± 2 45.1 90 ± 4 46.7 93.5 ± 0.1 200 199.8 99.9 ± 0.5 205.4 102.7 ± 0.2 198.9 99 ± 1 395.7 99 ± 2 402.7 100.7 ± 0.3 201.6 101 ± 3 500 500.4 100.1 ± 0.2 494.4 98.9 ± 0.1 500.7 100.2 ± 0.7 1001.4 100.3 ± 0.3 988.2 99 ± 1 498.2 99.7 ± 0.7 Eh vlue is the men of 3 replite etermintions. SD = stnr evition. Anlyte e t 400 mg/l. Anlyte e t 1000 mg/l. LC Proeure A10µL liquot of solution ontining the nlytes in their liner ynmi onentrtion rnges re injete into the liqui hromtogrph: 50 500 mg/l for SA, CF, PCT, n PY n 50 1000 mg/l for CO n TH. A flow rte of 1 ml/min n working olumn temperture of 35 C re use. The ompouns re seprte on reverse-phse C 18 Nuleosil olumn, with moile phse onsisting of the 2 suessive elunts esrie ove, oth juste to ph 2.1 with phosphori i efore the wter n etonitrile re mixe. After ll the ompouns re seprte, the wter is psse through the olumn for 4 min. Asorne pek res re mesure in ll ses. Results n Disussion Temperture n Flow Rte In the propose LC metho, the temperture vrition n the flow rte for the resolution of the system i not hve ny signifint influene on the nlytil signl. However, temperture of 35 C ws use euse it llowe liqui phse with lower visosity, n flow rte of 1 ml/min, whih ws pproprite for the working pressure of the hromtogrphi equipment, ws use to shorten the time require to perform the nlysis. Orer of Elution Preliminry stuies with severl elunt systems were onute to selet the most effetive elunt for the seprtion of the 6 nlytes of the system. With some elunts, wter t ph 5.0 n etonitrile wter from (25 + 75, v/v) to (75 + 25, v/v), lso t ph 5, no resolution ws oserve euse of lrge overlp of the signls (for exmple, the CO n PY peks). Moreover, symmetri n very wie peks were otine for wter-solule vitmins. With more ii elunt, etonitrile wter (75 + 25, v/v), ph 2.1, thinner peks s well s goo seprtion were hieve for CO with respet to wter-solule vitmins, ut PY n TH were not stisftorily seprte. With only wter t ph 2.1, PY n TH were seprte stisftorily, ut the other 4 ompouns, with lower polrity, were not elute. It oul e expete tht the use of less polr moile phse fter the elution of the wter-solule vitmins woul seprte them. Methnol wter (35 + 65, v/v) ws trie, ut it file to proue goo seprtion for CO n PCT. Finlly, etonitrile wter (75 + 25, v/v), ph 2.1, gve stisftory resolution of the 4 ompouns. Therefore, the moile phse selete for the most effiient seprtion onsiste of 2 elunts: the first elunt ws wter (LC) juste to ph 2.1 with phosphori i. It llowe the seprtion of the vitmins, whih were retine more wekly in the polr sttionry phse euse of their ioni nture. The seon elunt ws etonitrile wter (75 + 25, v/v), lso juste to the sme ph with phosphori i, for the seprtion of the 4 remining omponents of the system. When the elution time for the first elunt ws inrese from 3 to 5 min, the signl from PY ws gretly improve, prouing only 1 pek (elution times of <5 min gve 2 lose peks). This vrile, however, h no in-
680 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Tle 5. Results for the etermintion of etylsliyli i, ffeine, oeine, n pretmol in ommeril phrmeutil preprtions Aetylsliyli i Cffeine Coeine Pretmol Phrmeutil preprtion Avg. foun ± SD, Avg. foun ± SD, Avg. foun ± SD, Avg. foun ± SD, Anlgils 30.0 27 ± 2 10.0 10 ± 1 500 458 ± 1 Dolmen 500 486 ± 3 10.0 10.5 ± 0.2 Dolvirán 400 387 ± 3 50.0 46 ± 1 9.6 9.4 ± 0.2 Fiorinl 200 197 ± 3 40.0 31.1 ± 0.4 14.7 14.6 ± 0.2 300 274 ± 4 Rinomiine 30.0 27.7 ± 0.7 150 138 ± 1 Eh vlue is the men of 3 replite etermintions. SD = stnr evition. fluene on the resolution of the other ompouns, inluing TH. With the seon elunt, 9 min ws shown to e pproprite to otin stisftory resolution (Tle 1). A typil hromtogrm is shown in Figure 1. Before the next injetion the olumn ws reequilirte y pssing ultrpure wter through the olumn for 4 min. This ws neessry to return the olumn to onitions pproprite for seprtion of the wter-solule vitmins. Only fter this proeure were the peks of the vitmins resolve. Clirtion n Anlytil Prmeters Stnr lirtion grphs for the nlytes were onstrute y plotting pek res proue y injetion of stnr solutions in the following onentrtion rnges: 50 500 mg/l for SA, CF, PCT, n PY, n 50 1000 mg/l for CO n TH. Clirtion urves were nlyze y regression nlysis, n the orreltion oeffiient (r), slope, n y-interept for eh run were lulte (Tle 2). This proess ws repete 3 times for sttistil nlysis. The etetion limits (DL) for eh ompoun, lulte oring to the riteri of Miller n Miller (21) n Curos et l. (22), s well s the liner ynmi rnges (LDR) n reltive stnr evition (RSD) t 2 nlyte levels re shown in Tle 3. Applitions The propose metho ws use in the simultneous etermintion of the nlytes in oth syntheti mixtures n ommeril phrmeutil formultions. Syntheti mixtures. Solutions ontining the 6 tive priniples t 3 ifferent levels were prepre n nlyze y the propose metho. Three etermintions were performe in ll ses. Results foun were ompletely stisftory (Tle 4). Commeril phrmeutil preprtions. Thirteen ommeril phrmeutils were nlyze y the evelope Tle 6. Results for the etermintion of oeine, pyrioxine, n thimine in ommeril phrmeutil preprtions Coeine Pyrioxine Thimine (NO 3 or HCl) Phrmeutil preprtion Avg. foun ± SD, Avg. foun ± SD, Avg. foun ± SD, Benom 300 277 ± 3 Coeisán 28.7 26.1 ± 0.5 Conuts 30.7 29.9 ± 0.4 Nervoión 100 103 ± 2 100 114.4 ± 0.3 Neurovur 250 237 ± 6 250 241 ± 3 Pzronquil 1.00 1.00 ± 0.01 0.60 0.6 ± 0.1 Perurets e oeín 50.0 46.1 ± 0.4 Serfoxie 300 280 ± 3 Eh vlue is the men of 3 replite etermintion. SD = stnr evition. Expresse s pyrioxine α-etoglutrte. Expresse s nhyre pyrioxine phosphoserinte.
Tle 7. Reovery of sliyli i, ffeine, pyrioxine, thimine, oeine, n pretmol from fortifie ommeril phrmeutil preprtions Sliyli i Cffeine Pyrioxine Thimine Coeine Pretmol Phrmeutil preprtion Anlgils 50 99 ± 1 100 101 ± 4 50 100 ± 2 100 105 ± 2 200 98 ± 4 200 98 ± 2 350 96.2 ± 0.2 300 98 ± 4 400 100 ± 1 Dolmen 20 98 ± 1 100 101 ± 1 50 99 ± 2 200 102 ± 1 100 99.4 ± 0.6 300 99.3 ± 0.7 Dovirán 50 101.4 ± 0.3 50 98 ± 1 100 99 ± 2 100 100 ± 1 100 101.7 ± 0.6 200 98 ± 2 150 99.3 ± 0.4 350 102.4 ± 0.5 300 99 ± 1 Fiorinl 50 98.4 ± 0.7 50 98 ± 1 100 98.4 ± 0.5 100 101.2 ± 0.3 200 99 ± 1 200 100 ± 1 150 101 ± 1 300 98.4 ± 0.6 300 102 ± 2 Nervoión 60 98.8 ± 0.2 60 99 ± 4 200 105 ± 2 200 105.5 ± 0.9 400 99 ± 2 800 99 ± 1 Neurovur 60 101 ± 2 60 102 ± 5 200 100.8 ± 0.9 200 104 ± 2 400 99 ± 1 800 97 ± 3 Pzronquil 100 92.6 ± 0.7 100 95 ± 1 200 101.1 ± 0.3 200 96 ± 1 300 104.6 ± 0.6 300 97 ± 1 Rinomiine 150 98 ± 1 400 95.5 ± 0.6 200 97 ± 2 800 96 ± 2 300 95 ± 2 1200 95 ± 2 Eh vlue is the men of 3 replite etermintions. SD = stnr evition. RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 681
682 RAMOS-MARTOS ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Conlusions The propose metho llowe suessful seprtion n etermintion of 6 tive priniples: 1 orgni i (SA), 2 lklois (CF n CO), 2 wter-solule vitmins (PY n TH), n phenol erivtive (PCT). The totl time require for the nlysis is reltively shorter thn tht esrie y other reserhers (8, 9, 13) for simpler systems ontining some of these nlytes (ut not ll). Its pplition to phrmeutils fter vlition emonstrte tht the propose metho n e use stisftorily for the etermintion of these nlytes. We ompre the LC proeures propose y the Unite Sttes Phrmopeil Convention (USP; 23) with the evelope metho esrie here. The RSDs of the results otine y the USP methos n this metho re similr; however, the moile phse of this metho is simpler, n lthough the tiling ftors, in generl, re similr, the resolution otine with this metho is etter. In ition, the metho propose in this pper inlues more ompouns; the USP methos for CO usully inlue nother nlgesi (suh s PCT or ASA, ut not oth simultneously), n in no se o they inlue wter-solule vitmins. Aknowlegment We knowlege the finnil support from the Ministerio e Euión y Cultur e Espñ (Direión Generl e Enseñnz Superior e Investigión Científi), projet PB 97-0849. Referenes Figure 2. Typil liqui hromtogrm otine y the evelope metho n showing the seprtion of the 4 ompouns from Fiorinl psules. Conitions: etetor, 285 nm. Peks: 1=PCT;2=CO;3=CF;4= SA; n lost pek = sliylmie. metho fter issolution n suitle ilution s inite in the setion on smple tretment. Results n lel lims y the mnufturer re summrize in Tles 5 n 6. In ition, s hek on the ury of the propose metho, reovery stuy ws performe in whih the respetive tive priniples ontine in severl phrmeutil preprtions were e t 3 levels. Goo reoveries were otine in ll ses (Tle 7). Figure 2 shows the seprtion of the 4 ompouns (PCT, CO, CF, n SA) from Fiorinl psules. (1) Aler, L.L., Overton, M.M., & Smith, D.E. (1971) J. Asso. Off. Anl. Chem. 54, 620 626 (2) Oesh, M., & Shli, M. (1974) Phrm. At Helv. 49, 317 322 (3) Auirjeie, A.M., Ael-Hmi, M.E., & Irhim, A. (1989) Anl. Lett. 22, 365 375 (4) Pirol, R., Breggi, S.R., & De Beneitis, G. (1998) J. Chromtogr. B Appl. 705, 309 315 (5) El-Shnwny, A., El-Sek, M., Aoul-Khier, A., & Ruker, G. (1991) J. Phrm. Si. 53, 209 212 (6) Siso, W.R., Rittenhouse, C.T., & Everhrt, L.A. (1985) J. Chromtogr. 348, 253 260 (7) Siso, W.R., Rittenhouse, C.T., Everhrt, L.A., & MLughin, A.M. (1986) J. Chromtogr. 354, 355 362 (8) El-Kommos, M.E., & Emr, K.M. (1988) Tlnt 36, 678 679 (9) Gámiz-Gri, L., & Luque e Cstro, M.D. (1997) J. Liq. Chromtogr. Relt. Tehnol. 20, 2123 2133 (10) Ppoynnis, I.N., & Cy, B. (1987) Mirohem. J. 36, 182 191 (11) Atwell, S.H., Bell, R.G., & Vys, R. (1992) Phrmopeil Forum 18, 3735 3745 (12) Sntoni, G., Fi, L., Grtteri, P., Renzi G., & Pinzuti, S. (1992) Int. J. Phrm. 80, 263 266 (13) Verm, K.K., Snghi, S.K., Jin, A., & Gupt, D. (1987) J. Phrm. Si. 76, 551 553
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