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__ bUMMARY
.-
A svey of the literature h& been carried out, covering pharmaceutical applititioks of high-performance liquid chromatography (HPLC) from 19724975. The antibiotics, nitrogen&bstra&s are divided into sevea groups, vi,. alkaloids, coniainihg compounds, steroid, sulphur-containing- compounds, fbrmdutions, and gesieral analytical techniques. A general ?esumC of the development of HPLC in our _.own labor&tory from 1969 onwards is given together with fourteen cbromatograms kovekis&the spectrum of our more novel activitik in pharmaceutical analysis. e INTRODUCTION The 1968 Medicines AU IegislatiOn in Great Britain, together with similar iegislation- in the rest of the world, led to a further tightening of the screws on the production and control of pharma&uticak. More searching questions than ever before began to be asked about the control of impurities in starting materials, intermediates, and bulk medicioals. As a direct result this, analysts in the pharmaceutical industry bad to search for more spe&ic and also mote-sensitive methods of analysis than ever before. They turned to : gas-liquid cbrotiatography (GLC), thin-layer cbromatography (TLC) and Liquid tihromatography (IX). TLC and LC were not readily acceptable because of tk. difsculty -in quantifying T&C and because of-the slow and inefkient nature of LC.-It is therefore ti& surpris@g$bat GLC bore the main brunt ofthe burden ‘of-increase in- sti$fe atibers; and every conceivabl& method- was adopted in order to _+ake these cOmplex ph~ceutical mofecuks volatile. However, with certain. &ou@ ‘&f-&mtiou&ds, e.g., cor+i+t&ids’ and antibi+i&, the use Of GLC was impossible atid. on& had to resort entirety fo EC. Figs. f and 2 iliusQate typical cbromato&$.ms d@nt$ by the &tiek method, clea&y demonstitig its inefkierzy and slowness_ _- .:.; -,.
of
.. I&% 1969 saw the impoe-of the .&st commercial liquid chromatograpbs from. :ihi :&~&+~~St&es and, after & first- cfiromat&rani of .a steroid mixti*e had. been ru& th& po&ntid of the_k&.nique in’ph&mac&uticaf. analysiS-was understood- The : $emn@ ma&&&s fo&nd ~&ems&es in at. t&s :stage- was that fhe o&y :inskkents $tiilabEe were-.&& Iar& .search --type, ;costing ElO,OW-l2,OW, so when the first
Voiume
of eiuant
hi)
FIg.1.Cunresrelatingabsorix1~1ce volumefor~u~~oloneace~~deandotherfurrentIya~able +pred-. COrtkosterokis, ----,Betan~etbsone 17-v&r&e; --em--.bydroc&isone acetate; 3uocinobne wotide. nisobne auztate; ------, txiardnolone acetonide;-,
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75
_Thik pauc@y of pubkati&s can be exp&ted to. change very rapidly with ‘the;ever _in&~&g availability of commer&I &strumentation, with improved &I~ k&oIogy and with-the reafisation that many separations which had hereto--.fore bee&both tedious and compI.kated, may now be effected quite readily by means of this powerful technique”. More surprising observations from- our own point of view are: (I) The very small number of contributiotis or&inating from Great Britain, Z.e..12 “A_(2) The very small number of papers concerned with quantitative analysis which report such important pa&ret&s to the analyst as reproducibility, standard error, etc. (3) The even smaller number of publications &so than 5%) recording variable-wavelength monitors for use at wavelengths other than 254 nm and the utihsation of the benetits which this presents of increased specificity and sensitivity and in many cases the added bonus of a reduction of interference from excipients. PEXARMACEUTKAL
APPLIcAlloNS
dlkahids
The applicability of HPLC for the analysis of alkaloids is probably one of the best recorded; abut sixteen papers have been published so fa#-lg, varying in their applicabibty from the analjks of plant extracts to the quantitative analysis of multicomponent drug products. The columns used appear almost equally divided between pellicular packings, e.g., Zipax and C&as& and ion exchangers, e.g. SCX, WAX, and modified polystyrenes. Anfibiotics
The work in this field has been dominated by the examination of tetracycline and its derivatives. Antibiotics are produced by fermentation processes -most of the standard methods of analysis are mierobiologieal. These procedures are useful for measuring activity against a specific test organism but do not tell the analyst anything about the chemica! purity of the medicinal. On the basis of these tests it is di%icult to carry out any process development work to increase the purity or yield of the product. Nine papers have so far been published20-28, using a wide variety of adsorbants, e.g., Kieselguhr, ion exchangers, and pellicular packings, e.g., Zipax, HCP, and Bondapah csS but almost exclusively eluting with some form of buffer system. Nitrogen-con faicing compounds This should be a popular area for exploitation
in pharmaceutical analysis. ContainedS in this group are the tranquilhsers: benzodiazepines, butyrophenones, earbamates, phenothiazines, thioxanthenes, etc. Because of the basic nature of many of the nitrogen-containing compounds, eluant phases in this group often contain bases; e-g., ammonia, ðyl&e; the complete Lange of column packings are recorded from ion exchangers throughpellic&r packings to adsorbants. Nineteen references to separations involving nitrogen-containing compounds have been found2947 _~
This was real&the area where the true potent&&lof HPLC in pharmaautkl analysis~ was .- first see& Derivatisation followed by GLC.was extremely difkult for
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this class of.;compounds in general and-impossible. for cort&%teroids in particirlar. Steroids are, in genera& potent compounds and as-such are formulated in iow-dosage formulations, hence the analyst is not’only-faced with sninvolatile compound -but with only a very small amount of it-in-a Iformulation, usually a tables a cream, or -an ointment.. Undoubtedly the best way to tackle a cream or. ointment is bjr HPLC. Much of the early work in this. field was carried out on pellicular-~colunins ~&r&y with reversed-phase eluants- Of late the pattern has been changing to tid&rbants with organic eluant phases. Eleven references(s-58 are given to work carried out in this field to date. Su!pht.ir-containing compounds ‘This is an important group of compounds, confainiug the sulphonanides. Most of the published separations5g66 are on ion exchangers tith btier~ solution eluants. Lroi-muhtions
HPLC really makes its greatest contribution in this area:, Because the complete analysis is carried out in the liquid phase -either aqueous or non-aqueousthe ex’ traction of the desired constituent from a formulation can be developed in such a -way that the extracted material is miscible with the eluant. Because of the sensitivi~ of the technique, -smaller amounts of sample may be taken, which in turn reduces the interference from excipients. In many cases it is.su&ient to grind tablets with methanol, filter, and apply an aliquot of the extract to the column. With ointments it is often sufl’icient to .shake the formulation with a mixture of aqueous alcohol and isooctane in order to remove the fatty excipients from the drug. It is surprising on reviewing the literature to find so few references to the analysis of formulations67-?6. General techniques This section covers the whole field of pharmaceutical analysis, from a general review paper on applications”-82, dru g purity proGles’*, to. an excellent paper+ on quick identi&zation methods of accurate stability for active ingredients in analgesics. This paper is one of the few recorded which quote quantitative data cotipnring peak height and peak area measurements, and also tables on accuracy. Five papers were., founds5* which discuss the-use of fluorescence detectors, with special reference to pharmaceutical analysis. Two general papers were published studying.&lumns and elur&tsystems; the first, by Parr-is“, describes the use of ternary liquid systems containing .dichloromethane, methanoi, and water on the. adsorbant. Zorbax-Sil. The second, by Twitchettgl, ev&ates the use of octadecylsilane chemically bonded to. 10m silica (Bondapak C,,). The drugs selectd are eluted with aqrieous.{meth&ol zt different concentrations. Retention valr.resfor 30 compounds aregive& A review paper by Frei= discusses instrumental needs in pharmaceutical analysis, and ilhrstmtes this with applications to the separation of &&lo&,. vitamins,antibiotics~ barbiturates, and glycosides, The last paper describes the sophistication of couplinga liquid chroSatograph to a &ass spectrometer79 and illustrates its appli&ion to the Gparation _-..:..-. . and identi&ation~of three sulphon%nides.. : In Gre+t S&a&t since .1969 the developm&t if the ~&l&&e of LC has been 1. taking place in two distinct phases, -1963-11973saw’tiuch inst&ent development ._- in =
. -APPLICkTTONSOFRPLCENPHARh~~~~TfCALINDUSTIEY
77
andreliability and-fke i@&minabIe argument on constant pressureversus~constantffow has been gobi~~onto tkis day, Detectors tirereai!Iythe problem area in that we have no universa1detector. Tke detectorswkick have cEaimedto be tivers& are not sensitive enough and have nbt stood the test oftime when comparedto the W and refractive index detec&. fn the pkarmaceuticalindustrywe are fortunatein that the majaa-ity of.~compotids$roduced have some kind of W absorbance, and if the exttiction coefficientis as low as 20 at 220 nm, quantitativeanalysescan stillbe carriedout using the vtiable-wave!ength detector,%vkickwas introducedin early 1972. _ Table L clezly shows the variation in absorbance maxima in steroids. Tke ability to select the monitoringwaVelen,& offerstke followingadvantages:(2)greater sensitivity,(2) greaterspecificity,especidy if the impuritiesor degradationproducts do not absorb at that wavelength,_md (3) in many cases a reduction of interference from excipients. The developmentof new instrumentsseemed to lose impetus from 1973 on-wardsand in recentyearsthe efforthas beenontke developmentofcolumntecknology. For the firsttwo or threeyearsHPLC was carriedout almost exclusivelyon pellicular mater&, from I973 onwardsthe fashion has been to tum to adsorbants,especially t~~twb~~2?reasofpumpsand~et~tow.Pumpsya~~edinc~~pie~~
TABLE
I
ULTRAYiOLET
ABSORPTiON
OF CHROMOPHORIC
GROUPS
IN STEROIDS
Chromophores
Ubravfokt
Carbonyl
170-200 280-300
Double
180-225
204
3,300
230-270
253
11,200
232 239 248
21,500 23,.5ocl 16.000
262 271 282 293
7,700 11,400 I1*!300 6900
306
14,saO
284
=,aao
223 256
13,500 11,900 I5J#
bond
a$-Unsatura& Conjugated
ketone
absorph~on
dimes (different
rings)
Con&ate4
dienes (same
ring)
_Conjugated poiyenes
a3.’
~Fi&3. &ramat&am o$ a test &ture for &.&king the &cienq 05 honxS&&d rnicr&u-tiCuiate c-Ch&& ~&in, 10cm- x 4.6 r&n LID., pa&xi with 5-~msilka g&f; &lvent systems&bzxane-l% acetonitrile;~ pressurk~ 1Qi) p.s.i.; .%ow-rate, i ml/r&l; attenugtioa, 0.5 2z.u.f.a..1 =.Phenab+rcue; 2 = aeetophenone; 3 = nitrobenzene;4 = 2&dinitrotoluenc. Wavelength, 2.54 I&I. --.
Fig. 5. Cbromat~grarn of oxytetracycIine bulk drug. Co!umn, (20 cm x 4.6 mxnI.D.) packedwith 5-m LicbrosorbS&60; solventsysteq 0.03 M 3ZDTAd&odiumsalt + 0.03 M KHzPOc i 40% me+01 @H adjustedto 5.7 with0.1 M N&H); pressure,1500 p.si.; ffow-rate,0.6 ml/&; attenuation,0.2 a&Es.; wavelength, 263 mu. 1 = fl-Apooxytetracycline; 2 = a-apooxytetracycline; 3 = oxytetracycline; 4 = anhydroo%ytetra&cline; 5 = unknown. ~-
Fig_6: Cbroma@grarnof oxytetra~clineshowingthe inclusion of an internalstandard,sulphametl@ne. Column,(20~~ x 4.6 mm LD.) packed&th S-pmLichrosorb SMO; solvent system, 0.03 MEDTAdisodiumsalt t 0.03 MEZ&PO~ + ~%methanol(pHadjustedto 5.7with0.1 MNaOH); p&s!%e, II%@p.sli.; Bow-rate,0.6 sni/min;attenuation,0.2 a.u.f.s.; wavebngth,263 nm. 1 = fiApooxyt&acycEne;2 = suIpbamezatbine (internalstandard);3 = a-apooxytetracycline; 4 = oxytetracycline; 5 =a&&ooxyte&acycl&; 6 = unknown.
Fig_ 7 showkthe di&rence in sensitivityof the impurities in this mixture when monitored at 254 n.m rather than 273 mn. F&. 8 demonstrates the usefullnessof a doubleb&m speckphotometer with low-volume flow cells for “stop-fiow” spectra of peaks.’The chromatogzuq is run as normal. When an unidentifiedpeak appearson thechart the flow is “stopped at the top of the peak” and the spectrumof the trapped fraction scarmed__This information is invaluablewh~~rmming a formulatedsampie
Fii 7. Cbromatograms of substitutedpLzeooIicmixtuk demonstrating tk diE&ence i& respons& at 2.54and 273 nm. (a) Calunm, 33cm x 4.6 mm I.D., QZC!G&with S_lrm Spherisorb SSNsilica; pressure, 150 p.s.i.; attenuation, 0.05 a_u.Es.; flow-rate. 2 &-&nin; solvent system;*he $- 1O? titonitriie i- 0.2% ethanol; wavelength, 273 nm. (b) Same conditions~as(a);exckpt wak+ngtb. 254 nm;
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Ellal~ w&e I.& cq 13-1 chlorratiine plum-2%
Fi& 9. Scliematic diagram of ti automated high-pressure liquid chromatograph. .- - -, Signal wd *&pGter ties;:. . .: . ., vacuum lines; ------, pneumatic lines; ?, hydratic lines. gig. l@.‘&aph
demonstrakg linearity of chromatograph response to chlorhexidine.
e&act or a’degiaded szim& f&r the first tint& Inmatiy,+es jt-w$ tee you &C&&k J -obtained is from the active agent orfrom the:excipients. As~~t~td~@eviotis~y k’this paper, when a method has been developed--for -a.tie% $h&iaceutic& a&%an.a$zty time &f 10~15min has been achieved, sampfes appear.-from -aH~directio~.~process.. develo@ment, stability, formulaticn develoljmeI;t- and vej&onthe nm&ei e&q?ds that which is humanly possible-to-be arialysed by,‘one Orson durmg a-73fh day.:Tfie machine then has to be made to work iivemight, using automated~injection and so&eform of data handling. This procedure has been carried out for -theanalysis of chlbrhexidine=. The schematic system is. shown in i;ig. 9 and the results obtained are shown in .Figs.-N-12.
Fig. i2. Chromatograms of duplicate injeztions of chlorkexidines&t&a&i geft) and an extract of Savlon hahy lotion (right). Column (10 cm x 4.6 .mm I.D.) packed with 1 l-pm silica gei; soloent system, acetonitrile-O.02N sulphuric acid in water (9l.S:S.S); pressure,300 psi.; fiow-Me, 1 ml] ruin; attenuation, 0.1 atrt.f.s.; wavebgth, 254 run. First peaks are due to solveritor solveritand excipiez&. CONCJXWON
if asked to predict the. progress over the next five years, we would suggest 8 Continuatioti of-our presei~t cours$, Le. .devclopment of more ~@ermanent@bo&d stationary phases on silica and alumina; a more general accep*ticeof v$+b~e wave-, length, and a+--appreciation of the.contiibution of W. specti E$catiie.of the high cost:of Capital e@ipment more mileage .mu& be obtai&d out df’&isting ma&i&s thr&gh auto&ted inject& and data.handhixg-
1 F. Bailey; k Holbrwk and R J. hliikr, J_ Pkzrm_ Pfiarqz~coL., 18 (1966) 12s. z--F. Baiky, j. P+rm Pharmucoc., 21 (1969) 405. 3 A. F. Mic&elis and D. W. Co&&, 3. Pharm. Sci., 62 (1973) i399. 4 W.‘ D. Brendel;Phhrin. Ztg_, 118 cl97311583. 5 P. J1Cas@an and J; L Thornton, d Forexiic SC& 12 (1972) 417. 6 Q_ N. BeasIey and T. HI. S_ CharIes, 1. Pharm_ Sci_, 62 (1973) 1691. 7 J. N. Done tid J. H. Knox, Process. Biochem., 7 (1972) 11. 8 J. L. Frahn md R J. Ilknan, J. Chmmatagr..,87 (1973) 187, J. D. M&Neil and R. W. Frei, 6. Chromarogr., 77 (1973) 425. 9 R. A. H eauxk, K. R. La&k, 10 G. IL JoiliKe and E. J. Shelkd, .i_ C/zromoYogr., 81 (1973) 150. 11 J. H. Knox and J_ Jurand, J. Uwomarogr., 82 (1973) 398. 12 J. S. MayelI and C. F. Hishey, Anal. Chem., 46 (1974) 449. 13 E. M&ia, P. Richards and H. F. W&on, J. C/vom~?ogr.,87 (1973) 523. 14 J. 33.Smith and J. c, Mollka, Amer. Lab., 4 (i972) 13. 15 M. H. Stutz ad S..Sass, Anal. Chem., 45 (1973) 2134. 16 J. D. Withver and J. H. Kluckhohn, J. Cliromo~ogr.SC& 11 (1973) 1. 17 C. Y. Wu and S. Siggia, Anal. Chent., 44 (1972) 1499. 18 C. Y. Wu and S. Siggia, And Ch&. Acm, 63 (1973) 393. 19 T. H. Beasley and D. W_ Smith, L Ass. O&%z_And Chem., 57 (1974) 85. . 20 P. P. As&e, J. B. Zzgar and G. P. Chrekian, J. Chrumatogr., 65 (1972) 377. 21 A. Bracey, J_ Pharm SC& 62 (1973) 1695. 22 A. G. Butterfield and D. W. Hugh-, Anfimfcrob. Agents Chemother., 4 (1973) 11. 23 K. Tsuji and J. H. Robertson, AR& Chem., 46 (1974) 539. 24 W. Me&in&i and C. P. schatfner, J_ Ckomatogr., 99 (1974) 619. 25 K_ Tsuji and J. H. Robertson, J. Chromatogr., 94 (1974) 245. 26 Et Tsuji, J_ H_ Robertson and J. A_ Bach, J_ Chromatogr., 99 (1974) 597. 27 W. Mo~~~wich and R G. Williams, X Plrarm. SC& 64 (1975) 313. 28 J. H. Knox and J. Jurand, J. C/‘womarogr.,110 (1975) 103. 29 P_ J_ Casknn and J. I_ Thornton. J. Cfzromorogr.Ski.. 11 (1973) 7. 30 G. B. Cox, f. Chromatog., 83 (1973) 471. 31 A. J. Fa&, 1. Phurm. Sci., 63 (1974) 274. 32 G. Gauche1 and F. D. Gauche& Z. KIti. Cizem.Kiln. Bfochem., 11 (1973) 35. _ 33 T. lkaba and J. F. Brien, J_ Chromatogr., 80 (1973) 161. 34 A. Menyharth and F. P. h+hn, J. Pharm. Sci., 63 (1974) 430. 35 J. Merzhauser and E. Roeder, Klin. fT’ochenschr., 51 (1973) 883. 36 J. A. Molka and G. R. Padmanabham, Amzf. Chem., 45 (1973) 1859. 37 R W. Roes, J. P&urn. Sk, 61 (1972) 1979. 38 D. J. Weber, J. Pharm. Sci., 61 (1972) 1797. 39 D. H. Rogers, J. Chromatogrw ScL, 12 (1974) 742. 40 L. Hoi&erg and J. T. Stewartt J. Pharm. Sci., 64 (1975) 1201. 41 N. D. Brawn, R T. Lot%exgand T. P_ Gibson, J. Chromofogr., 99 (1974) 635. 42 -W. Ltidner, R. W. Frei and W. Santi, J. Chromatogr..,108 (1975) 299. ~43D.-Mo@s and C. K. Woa J. Pharm. SC&, 64 (1975) 123. 44 I. .D. W2tsc.m and AM.J_ Stew J. ciWOMQtOgrm,_ t 10 (1975) 389. 45 _B. Liadstriim, J. Chromatogr., 100 (1974) 189. 46 R_ M. R&gin and A. L. Schnidt, .i_ PErarm. Sci., 64 (1975) 680. ~47 E. Murgia, P. Richards and H. F. Walton, J. Chromofogr., 87 (1973) 523. 48 J. A. Molfica rind R. F. S&usz, & Bharm. Scf., 61 (1972) 444. 49 M. 6. Olson, J, Phtzrm. Sci., 62 (1973) 2001. 50 I: C. T&&stone and W. Wortrnanne J_~Chzomaro.gr_, 76 (1973) 244. 51 W. Workann, 6. sthnabel and J. C. Touchstone, J. Ciuomorogr., 84 (1973) 396. 52 F. Baiky .ar~d P. N. B&taiq i. Phprm. Pharmacot., 24 (1972) 425. 53 A. X Butte&i&d and B_ A. Lo&e, JI Ckomorogr. Sc& 11 @m_ al. 54 F. k ?&tzpatrkk and S.-S&& Am& Chen2..44(1972) 2211. 55 R A. Henry sd J. A.+S&nit, J. Chromafogr. Sci., 9 (1971) 513.
_.
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--..‘--...: _..: .-.: & G.. I&X&ield qnd -B.‘A; Lodge, I. P&m-m S+ 64 (iis’?-5) 4.411 ---- 1. : ‘57. -AL R. &&n&,~J(. Chmtiq&gr_, 92 (1974).465. .z ..- .:I-58 S: L. Sarkaqtid Et V. Jogi, J. f?hr&~r~~__S&.; 12 (1974) 206. ..- .-.-~:--. .: 59 T_. C. Kt$m, %Phtirm;~‘Sci.,- 61 (19TZj~254. --60 IX J. Neisbn, C_ J: L;:Bugg& H. C. EQzsny and T. @. Zin&x&&;~. Chrb&&r.~ 77(i@3jkl. 61 R.-B. Poet artd Hi H. &, J. Pi?ur& Ski.; 62 (197s) 809. ._ : ._ .,...-. -. .I_ 62 N. J,~Pound and. R_ .W. sears, C’ori;,J. Pkayt. Sci..,_S (1973) S+ 63 3; S. Wragg, Pbarm. J., 211 (1973) 565. -= _ : 64 G_ K Gordon 2nd D. C; Ghdul;:J. Ph&r. S&i., 64 (19751-1%. 55 T. Inaba, M. E. Besfey z@ E. 1; Chow, L.- Chromatu&, lU% (19%) ‘165. 66 B. L.. Karger and S .C. Su, i. Chrumafogr. Sci., 12.(1974)-.67S, 57.4; _Menyharth and F. P. Mann, _J. Pkarm; Sci.; 63 (19?4).430_ f. .: _ 33 R.-W. Roes, J.Phnrm. Sci., 61 (1972) 1979. 59 F. Bailey and P.. N. Brittain, J. Pfzarm. Phar~aVc& 24 (1972) 425.. -. 70 R.E. Heutteu+ir.an< A. P. Shriff, J. Chromatogk. Sci., 13 (1975) 357. 71 A. G. Butterfield and B. A. L@ge, .I. Pharm. Sci., 64 (1975) 441. : 72 W. C. Landgraf a&d E. C. Jennings; J. Pharm. Sci., 62 (1973) 278. 73 B. =-Penner, J’. Phkn. SC& 69 (1975) 1017. 74 L. L. Needham and-M; M. Kochhar, J. Chromatogr., iii .(1975) 422. 75 -A. P. De Leenheer,‘ S. S. Rigo, (3. F. 9elijktin.s and P. M:van Vaerenbergh, J. Chromatogr., 92 (1974) 339. 76 R. K. Gilpin, J. A. Kspi and C. A. Janicki, J. Chromzzfugr., -107 (197.5) 115.. 77 F. Bailey and P. N. Brittain, J. Chrumato&., 83 (1973) 431. 78 L. T. Grady and S. E. Hays, J. PhCmm_ Stii., 62 (1973) 456. 79 R E. Lovins and S. R. Eliis, Anal. Cbn~, 45 (1973) 1553. .80 M. Martin and G. Guiochon, Bull. Sac. Chim. Fr., (1973) 161. 81 J. S. Mayell and C. F. Hishey, Anal. Chem., 46 (1974) 449. 82 A. F. Michaelis and D. W. Comish, J_ Pirclrm. Sci., 62 (1973) 1399. 83l.S. G. Perry, Chem. EWr_, 7 (1971) 366. .84 T. M. Rajcsanyi and .L. Otvos, Separ_ Purif Metho& 2 (1973) 361. 85 R G. Muusze and J. F. K. Huber, J. Cirrotiiaiogr, Sci., 12 (1974) 779. 86 W- Lindner, R: W. Frei and W. Santi, J. Chromatogr.; 111. (1975) 365_ 87 J. F. Murray, Jr., G. R. Gordon, C. C. Gulledge and J. H; Peters, I. Chromatogr., 107 (1975) 67. 88~ W. Dunges and G. Naundorf, J. Chromarogr. Sci., I2 (i974) 655. 89 M. Roth.and A. Hunpai, J_ Chromatogr., 83 (1973) 353. 90 N. A. Parr& b. Chromatogr. Sci., 12 (1974) 753. 91 P. J. Twichett and A. C. Moeat, J. Chromazogr.. Ill (1975) 149. 92 R. W. Frei, ReS. Develop., 25 (E974) 32. 93 F. Bailey, P. N. Brittain and B. F. Williamson, J. Chromatigr., 109 (19%) 305. .-
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_+%ULEY ~mj&tS flYhem-&.Ltitrti fiehi ‘Cd~&)
LiMte& Pharqacekcak DivKm, Htm&fieU Industrial&tale, Maccies-
__ bUMMARY
.-
A svey of the literature h& been carried out, covering pharmaceutical applititioks of high-performance liquid chromatography (HPLC) from 19724975. The antibiotics, nitrogen&bstra&s are divided into sevea groups, vi,. alkaloids, coniainihg compounds, steroid, sulphur-containing- compounds, fbrmdutions, and gesieral analytical techniques. A general ?esumC of the development of HPLC in our _.own labor&tory from 1969 onwards is given together with fourteen cbromatograms kovekis&the spectrum of our more novel activitik in pharmaceutical analysis. e INTRODUCTION The 1968 Medicines AU IegislatiOn in Great Britain, together with similar iegislation- in the rest of the world, led to a further tightening of the screws on the production and control of pharma&uticak. More searching questions than ever before began to be asked about the control of impurities in starting materials, intermediates, and bulk medicioals. As a direct result this, analysts in the pharmaceutical industry bad to search for more spe&ic and also mote-sensitive methods of analysis than ever before. They turned to : gas-liquid cbrotiatography (GLC), thin-layer cbromatography (TLC) and Liquid tihromatography (IX). TLC and LC were not readily acceptable because of tk. difsculty -in quantifying T&C and because of-the slow and inefkient nature of LC.-It is therefore ti& surpris@g$bat GLC bore the main brunt ofthe burden ‘of-increase in- sti$fe atibers; and every conceivabl& method- was adopted in order to _+ake these cOmplex ph~ceutical mofecuks volatile. However, with certain. &ou@ ‘&f-&mtiou&ds, e.g., cor+i+t&ids’ and antibi+i&, the use Of GLC was impossible atid. on& had to resort entirety fo EC. Figs. f and 2 iliusQate typical cbromato&$.ms d@nt$ by the &tiek method, clea&y demonstitig its inefkierzy and slowness_ _- .:.; -,.
of
.. I&% 1969 saw the impoe-of the .&st commercial liquid chromatograpbs from. :ihi :&~&+~~St&es and, after & first- cfiromat&rani of .a steroid mixti*e had. been ru& th& po&ntid of the_k&.nique in’ph&mac&uticaf. analysiS-was understood- The : $emn@ ma&&&s fo&nd ~&ems&es in at. t&s :stage- was that fhe o&y :inskkents $tiilabEe were-.&& Iar& .search --type, ;costing ElO,OW-l2,OW, so when the first
Voiume
of eiuant
hi)
FIg.1.Cunresrelatingabsorix1~1ce volumefor~u~~oloneace~~deandotherfurrentIya~able +pred-. COrtkosterokis, ----,Betan~etbsone 17-v&r&e; --em--.bydroc&isone acetate; 3uocinobne wotide. nisobne auztate; ------, txiardnolone acetonide;-,
.-__..
-.’
‘.ti@j&&S
_.’- __ ._ . :.!@e Et&&&
OF HPw
IN PEIAEWkEtiCAL
--
iiZ&TR%
75
_Thik pauc@y of pubkati&s can be exp&ted to. change very rapidly with ‘the;ever _in&~&g availability of commer&I &strumentation, with improved &I~ k&oIogy and with-the reafisation that many separations which had hereto--.fore bee&both tedious and compI.kated, may now be effected quite readily by means of this powerful technique”. More surprising observations from- our own point of view are: (I) The very small number of contributiotis or&inating from Great Britain, Z.e..12 “A_(2) The very small number of papers concerned with quantitative analysis which report such important pa&ret&s to the analyst as reproducibility, standard error, etc. (3) The even smaller number of publications &so than 5%) recording variable-wavelength monitors for use at wavelengths other than 254 nm and the utihsation of the benetits which this presents of increased specificity and sensitivity and in many cases the added bonus of a reduction of interference from excipients. PEXARMACEUTKAL
APPLIcAlloNS
dlkahids
The applicability of HPLC for the analysis of alkaloids is probably one of the best recorded; abut sixteen papers have been published so fa#-lg, varying in their applicabibty from the analjks of plant extracts to the quantitative analysis of multicomponent drug products. The columns used appear almost equally divided between pellicular packings, e.g., Zipax and C&as& and ion exchangers, e.g. SCX, WAX, and modified polystyrenes. Anfibiotics
The work in this field has been dominated by the examination of tetracycline and its derivatives. Antibiotics are produced by fermentation processes -most of the standard methods of analysis are mierobiologieal. These procedures are useful for measuring activity against a specific test organism but do not tell the analyst anything about the chemica! purity of the medicinal. On the basis of these tests it is di%icult to carry out any process development work to increase the purity or yield of the product. Nine papers have so far been published20-28, using a wide variety of adsorbants, e.g., Kieselguhr, ion exchangers, and pellicular packings, e.g., Zipax, HCP, and Bondapah csS but almost exclusively eluting with some form of buffer system. Nitrogen-con faicing compounds This should be a popular area for exploitation
in pharmaceutical analysis. ContainedS in this group are the tranquilhsers: benzodiazepines, butyrophenones, earbamates, phenothiazines, thioxanthenes, etc. Because of the basic nature of many of the nitrogen-containing compounds, eluant phases in this group often contain bases; e-g., ammonia, ðyl&e; the complete Lange of column packings are recorded from ion exchangers throughpellic&r packings to adsorbants. Nineteen references to separations involving nitrogen-containing compounds have been found2947 _~
This was real&the area where the true potent&&lof HPLC in pharmaautkl analysis~ was .- first see& Derivatisation followed by GLC.was extremely difkult for
yi-
._
..
r.‘&.&y
. ..-
.’
this class of.;compounds in general and-impossible. for cort&%teroids in particirlar. Steroids are, in genera& potent compounds and as-such are formulated in iow-dosage formulations, hence the analyst is not’only-faced with sninvolatile compound -but with only a very small amount of it-in-a Iformulation, usually a tables a cream, or -an ointment.. Undoubtedly the best way to tackle a cream or. ointment is bjr HPLC. Much of the early work in this. field was carried out on pellicular-~colunins ~&r&y with reversed-phase eluants- Of late the pattern has been changing to tid&rbants with organic eluant phases. Eleven references(s-58 are given to work carried out in this field to date. Su!pht.ir-containing compounds ‘This is an important group of compounds, confainiug the sulphonanides. Most of the published separations5g66 are on ion exchangers tith btier~ solution eluants. Lroi-muhtions
HPLC really makes its greatest contribution in this area:, Because the complete analysis is carried out in the liquid phase -either aqueous or non-aqueousthe ex’ traction of the desired constituent from a formulation can be developed in such a -way that the extracted material is miscible with the eluant. Because of the sensitivi~ of the technique, -smaller amounts of sample may be taken, which in turn reduces the interference from excipients. In many cases it is.su&ient to grind tablets with methanol, filter, and apply an aliquot of the extract to the column. With ointments it is often sufl’icient to .shake the formulation with a mixture of aqueous alcohol and isooctane in order to remove the fatty excipients from the drug. It is surprising on reviewing the literature to find so few references to the analysis of formulations67-?6. General techniques This section covers the whole field of pharmaceutical analysis, from a general review paper on applications”-82, dru g purity proGles’*, to. an excellent paper+ on quick identi&zation methods of accurate stability for active ingredients in analgesics. This paper is one of the few recorded which quote quantitative data cotipnring peak height and peak area measurements, and also tables on accuracy. Five papers were., founds5* which discuss the-use of fluorescence detectors, with special reference to pharmaceutical analysis. Two general papers were published studying.&lumns and elur&tsystems; the first, by Parr-is“, describes the use of ternary liquid systems containing .dichloromethane, methanoi, and water on the. adsorbant. Zorbax-Sil. The second, by Twitchettgl, ev&ates the use of octadecylsilane chemically bonded to. 10m silica (Bondapak C,,). The drugs selectd are eluted with aqrieous.{meth&ol zt different concentrations. Retention valr.resfor 30 compounds aregive& A review paper by Frei= discusses instrumental needs in pharmaceutical analysis, and ilhrstmtes this with applications to the separation of &&lo&,. vitamins,antibiotics~ barbiturates, and glycosides, The last paper describes the sophistication of couplinga liquid chroSatograph to a &ass spectrometer79 and illustrates its appli&ion to the Gparation _-..:..-. . and identi&ation~of three sulphon%nides.. : In Gre+t S&a&t since .1969 the developm&t if the ~&l&&e of LC has been 1. taking place in two distinct phases, -1963-11973saw’tiuch inst&ent development ._- in =
. -APPLICkTTONSOFRPLCENPHARh~~~~TfCALINDUSTIEY
77
andreliability and-fke i@&minabIe argument on constant pressureversus~constantffow has been gobi~~onto tkis day, Detectors tirereai!Iythe problem area in that we have no universa1detector. Tke detectorswkick have cEaimedto be tivers& are not sensitive enough and have nbt stood the test oftime when comparedto the W and refractive index detec&. fn the pkarmaceuticalindustrywe are fortunatein that the majaa-ity of.~compotids$roduced have some kind of W absorbance, and if the exttiction coefficientis as low as 20 at 220 nm, quantitativeanalysescan stillbe carriedout using the vtiable-wave!ength detector,%vkickwas introducedin early 1972. _ Table L clezly shows the variation in absorbance maxima in steroids. Tke ability to select the monitoringwaVelen,& offerstke followingadvantages:(2)greater sensitivity,(2) greaterspecificity,especidy if the impuritiesor degradationproducts do not absorb at that wavelength,_md (3) in many cases a reduction of interference from excipients. The developmentof new instrumentsseemed to lose impetus from 1973 on-wardsand in recentyearsthe efforthas beenontke developmentofcolumntecknology. For the firsttwo or threeyearsHPLC was carriedout almost exclusivelyon pellicular mater&, from I973 onwardsthe fashion has been to tum to adsorbants,especially t~~twb~~2?reasofpumpsand~et~tow.Pumpsya~~edinc~~pie~~
TABLE
I
ULTRAYiOLET
ABSORPTiON
OF CHROMOPHORIC
GROUPS
IN STEROIDS
Chromophores
Ubravfokt
Carbonyl
170-200 280-300
Double
180-225
204
3,300
230-270
253
11,200
232 239 248
21,500 23,.5ocl 16.000
262 271 282 293
7,700 11,400 I1*!300 6900
306
14,saO
284
=,aao
223 256
13,500 11,900 I5J#
bond
a$-Unsatura& Conjugated
ketone
absorph~on
dimes (different
rings)
Con&ate4
dienes (same
ring)
_Conjugated poiyenes
a3.’
~Fi&3. &ramat&am o$ a test &ture for &.&king the &cienq 05 honxS&&d rnicr&u-tiCuiate c-Ch&& ~&in, 10cm- x 4.6 r&n LID., pa&xi with 5-~msilka g&f; &lvent systems&bzxane-l% acetonitrile;~ pressurk~ 1Qi) p.s.i.; .%ow-rate, i ml/r&l; attenugtioa, 0.5 2z.u.f.a..1 =.Phenab+rcue; 2 = aeetophenone; 3 = nitrobenzene;4 = 2&dinitrotoluenc. Wavelength, 2.54 I&I. --.
Fig. 5. Cbromat~grarn of oxytetracycIine bulk drug. Co!umn, (20 cm x 4.6 mxnI.D.) packedwith 5-m LicbrosorbS&60; solventsysteq 0.03 M 3ZDTAd&odiumsalt + 0.03 M KHzPOc i 40% me+01 @H adjustedto 5.7 with0.1 M N&H); pressure,1500 p.si.; ffow-rate,0.6 ml/&; attenuation,0.2 a&Es.; wavelength, 263 mu. 1 = fl-Apooxytetracycline; 2 = a-apooxytetracycline; 3 = oxytetracycline; 4 = anhydroo%ytetra&cline; 5 = unknown. ~-
Fig_6: Cbroma@grarnof oxytetra~clineshowingthe inclusion of an internalstandard,sulphametl@ne. Column,(20~~ x 4.6 mm LD.) packed&th S-pmLichrosorb SMO; solvent system, 0.03 MEDTAdisodiumsalt t 0.03 MEZ&PO~ + ~%methanol(pHadjustedto 5.7with0.1 MNaOH); p&s!%e, II%@p.sli.; Bow-rate,0.6 sni/min;attenuation,0.2 a.u.f.s.; wavebngth,263 nm. 1 = fiApooxyt&acycEne;2 = suIpbamezatbine (internalstandard);3 = a-apooxytetracycline; 4 = oxytetracycline; 5 =a&&ooxyte&acycl&; 6 = unknown.
Fig_ 7 showkthe di&rence in sensitivityof the impurities in this mixture when monitored at 254 n.m rather than 273 mn. F&. 8 demonstrates the usefullnessof a doubleb&m speckphotometer with low-volume flow cells for “stop-fiow” spectra of peaks.’The chromatogzuq is run as normal. When an unidentifiedpeak appearson thechart the flow is “stopped at the top of the peak” and the spectrumof the trapped fraction scarmed__This information is invaluablewh~~rmming a formulatedsampie
Fii 7. Cbromatograms of substitutedpLzeooIicmixtuk demonstrating tk diE&ence i& respons& at 2.54and 273 nm. (a) Calunm, 33cm x 4.6 mm I.D., QZC!G&with S_lrm Spherisorb SSNsilica; pressure, 150 p.s.i.; attenuation, 0.05 a_u.Es.; flow-rate. 2 &-&nin; solvent system;*he $- 1O? titonitriie i- 0.2% ethanol; wavelength, 273 nm. (b) Same conditions~as(a);exckpt wak+ngtb. 254 nm;
..
: :. .&I
Ellal~ w&e I.& cq 13-1 chlorratiine plum-2%
Fi& 9. Scliematic diagram of ti automated high-pressure liquid chromatograph. .- - -, Signal wd *&pGter ties;:. . .: . ., vacuum lines; ------, pneumatic lines; ?, hydratic lines. gig. l@.‘&aph
demonstrakg linearity of chromatograph response to chlorhexidine.
e&act or a’degiaded szim& f&r the first tint& Inmatiy,+es jt-w$ tee you &C&&k J -obtained is from the active agent orfrom the:excipients. As~~t~td~@eviotis~y k’this paper, when a method has been developed--for -a.tie% $h&iaceutic& a&%an.a$zty time &f 10~15min has been achieved, sampfes appear.-from -aH~directio~.~process.. develo@ment, stability, formulaticn develoljmeI;t- and vej&onthe nm&ei e&q?ds that which is humanly possible-to-be arialysed by,‘one Orson durmg a-73fh day.:Tfie machine then has to be made to work iivemight, using automated~injection and so&eform of data handling. This procedure has been carried out for -theanalysis of chlbrhexidine=. The schematic system is. shown in i;ig. 9 and the results obtained are shown in .Figs.-N-12.
Fig. i2. Chromatograms of duplicate injeztions of chlorkexidines&t&a&i geft) and an extract of Savlon hahy lotion (right). Column (10 cm x 4.6 .mm I.D.) packed with 1 l-pm silica gei; soloent system, acetonitrile-O.02N sulphuric acid in water (9l.S:S.S); pressure,300 psi.; fiow-Me, 1 ml] ruin; attenuation, 0.1 atrt.f.s.; wavebgth, 254 run. First peaks are due to solveritor solveritand excipiez&. CONCJXWON
if asked to predict the. progress over the next five years, we would suggest 8 Continuatioti of-our presei~t cours$, Le. .devclopment of more ~@ermanent@bo&d stationary phases on silica and alumina; a more general accep*ticeof v$+b~e wave-, length, and a+--appreciation of the.contiibution of W. specti E$catiie.of the high cost:of Capital e@ipment more mileage .mu& be obtai&d out df’&isting ma&i&s thr&gh auto&ted inject& and data.handhixg-
1 F. Bailey; k Holbrwk and R J. hliikr, J_ Pkzrm_ Pfiarqz~coL., 18 (1966) 12s. z--F. Baiky, j. P+rm Pharmucoc., 21 (1969) 405. 3 A. F. Mic&elis and D. W. Co&&, 3. Pharm. Sci., 62 (1973) i399. 4 W.‘ D. Brendel;Phhrin. Ztg_, 118 cl97311583. 5 P. J1Cas@an and J; L Thornton, d Forexiic SC& 12 (1972) 417. 6 Q_ N. BeasIey and T. HI. S_ CharIes, 1. Pharm_ Sci_, 62 (1973) 1691. 7 J. N. Done tid J. H. Knox, Process. Biochem., 7 (1972) 11. 8 J. L. Frahn md R J. Ilknan, J. Chmmatagr..,87 (1973) 187, J. D. M&Neil and R. W. Frei, 6. Chromarogr., 77 (1973) 425. 9 R. A. H eauxk, K. R. La&k, 10 G. IL JoiliKe and E. J. Shelkd, .i_ C/zromoYogr., 81 (1973) 150. 11 J. H. Knox and J_ Jurand, J. Uwomarogr., 82 (1973) 398. 12 J. S. MayelI and C. F. Hishey, Anal. Chem., 46 (1974) 449. 13 E. M&ia, P. Richards and H. F. W&on, J. C/vom~?ogr.,87 (1973) 523. 14 J. 33.Smith and J. c, Mollka, Amer. Lab., 4 (i972) 13. 15 M. H. Stutz ad S..Sass, Anal. Chem., 45 (1973) 2134. 16 J. D. Withver and J. H. Kluckhohn, J. Cliromo~ogr.SC& 11 (1973) 1. 17 C. Y. Wu and S. Siggia, Anal. Chent., 44 (1972) 1499. 18 C. Y. Wu and S. Siggia, And Ch&. Acm, 63 (1973) 393. 19 T. H. Beasley and D. W_ Smith, L Ass. O&%z_And Chem., 57 (1974) 85. . 20 P. P. As&e, J. B. Zzgar and G. P. Chrekian, J. Chrumatogr., 65 (1972) 377. 21 A. Bracey, J_ Pharm SC& 62 (1973) 1695. 22 A. G. Butterfield and D. W. Hugh-, Anfimfcrob. Agents Chemother., 4 (1973) 11. 23 K. Tsuji and J. H. Robertson, AR& Chem., 46 (1974) 539. 24 W. Me&in&i and C. P. schatfner, J_ Ckomatogr., 99 (1974) 619. 25 K_ Tsuji and J. H. Robertson, J. Chromatogr., 94 (1974) 245. 26 Et Tsuji, J_ H_ Robertson and J. A_ Bach, J_ Chromatogr., 99 (1974) 597. 27 W. Mo~~~wich and R G. Williams, X Plrarm. SC& 64 (1975) 313. 28 J. H. Knox and J. Jurand, J. C/‘womarogr.,110 (1975) 103. 29 P_ J_ Casknn and J. I_ Thornton. J. Cfzromorogr.Ski.. 11 (1973) 7. 30 G. B. Cox, f. Chromatog., 83 (1973) 471. 31 A. J. Fa&, 1. Phurm. Sci., 63 (1974) 274. 32 G. Gauche1 and F. D. Gauche& Z. KIti. Cizem.Kiln. Bfochem., 11 (1973) 35. _ 33 T. lkaba and J. F. Brien, J_ Chromatogr., 80 (1973) 161. 34 A. Menyharth and F. P. h+hn, J. Pharm. Sci., 63 (1974) 430. 35 J. Merzhauser and E. Roeder, Klin. fT’ochenschr., 51 (1973) 883. 36 J. A. Molka and G. R. Padmanabham, Amzf. Chem., 45 (1973) 1859. 37 R W. Roes, J. P&urn. Sk, 61 (1972) 1979. 38 D. J. Weber, J. Pharm. Sci., 61 (1972) 1797. 39 D. H. Rogers, J. Chromatogrw ScL, 12 (1974) 742. 40 L. Hoi&erg and J. T. Stewartt J. Pharm. Sci., 64 (1975) 1201. 41 N. D. Brawn, R T. Lot%exgand T. P_ Gibson, J. Chromofogr., 99 (1974) 635. 42 -W. Ltidner, R. W. Frei and W. Santi, J. Chromatogr..,108 (1975) 299. ~43D.-Mo@s and C. K. Woa J. Pharm. SC&, 64 (1975) 123. 44 I. .D. W2tsc.m and AM.J_ Stew J. ciWOMQtOgrm,_ t 10 (1975) 389. 45 _B. Liadstriim, J. Chromatogr., 100 (1974) 189. 46 R_ M. R&gin and A. L. Schnidt, .i_ PErarm. Sci., 64 (1975) 680. ~47 E. Murgia, P. Richards and H. F. Walton, J. Chromofogr., 87 (1973) 523. 48 J. A. Molfica rind R. F. S&usz, & Bharm. Scf., 61 (1972) 444. 49 M. 6. Olson, J, Phtzrm. Sci., 62 (1973) 2001. 50 I: C. T&&stone and W. Wortrnanne J_~Chzomaro.gr_, 76 (1973) 244. 51 W. Workann, 6. sthnabel and J. C. Touchstone, J. Ciuomorogr., 84 (1973) 396. 52 F. Baiky .ar~d P. N. B&taiq i. Phprm. Pharmacot., 24 (1972) 425. 53 A. X Butte&i&d and B_ A. Lo&e, JI Ckomorogr. Sc& 11 @m_ al. 54 F. k ?&tzpatrkk and S.-S&& Am& Chen2..44(1972) 2211. 55 R A. Henry sd J. A.+S&nit, J. Chromafogr. Sci., 9 (1971) 513.
_.
84 ..:-._ F-:
I
B$i$Y . . .
--..‘--...: _..: .-.: & G.. I&X&ield qnd -B.‘A; Lodge, I. P&m-m S+ 64 (iis’?-5) 4.411 ---- 1. : ‘57. -AL R. &&n&,~J(. Chmtiq&gr_, 92 (1974).465. .z ..- .:I-58 S: L. Sarkaqtid Et V. Jogi, J. f?hr&~r~~__S&.; 12 (1974) 206. ..- .-.-~:--. .: 59 T_. C. Kt$m, %Phtirm;~‘Sci.,- 61 (19TZj~254. --60 IX J. Neisbn, C_ J: L;:Bugg& H. C. EQzsny and T. @. Zin&x&&;~. Chrb&&r.~ 77(i@3jkl. 61 R.-B. Poet artd Hi H. &, J. Pi?ur& Ski.; 62 (197s) 809. ._ : ._ .,...-. -. .I_ 62 N. J,~Pound and. R_ .W. sears, C’ori;,J. Pkayt. Sci..,_S (1973) S+ 63 3; S. Wragg, Pbarm. J., 211 (1973) 565. -= _ : 64 G_ K Gordon 2nd D. C; Ghdul;:J. Ph&r. S&i., 64 (19751-1%. 55 T. Inaba, M. E. Besfey z@ E. 1; Chow, L.- Chromatu&, lU% (19%) ‘165. 66 B. L.. Karger and S .C. Su, i. Chrumafogr. Sci., 12.(1974)-.67S, 57.4; _Menyharth and F. P. Mann, _J. Pkarm; Sci.; 63 (19?4).430_ f. .: _ 33 R.-W. Roes, J.Phnrm. Sci., 61 (1972) 1979. 59 F. Bailey and P.. N. Brittain, J. Pfzarm. Phar~aVc& 24 (1972) 425.. -. 70 R.E. Heutteu+ir.an< A. P. Shriff, J. Chromatogk. Sci., 13 (1975) 357. 71 A. G. Butterfield and B. A. L@ge, .I. Pharm. Sci., 64 (1975) 441. : 72 W. C. Landgraf a&d E. C. Jennings; J. Pharm. Sci., 62 (1973) 278. 73 B. =-Penner, J’. Phkn. SC& 69 (1975) 1017. 74 L. L. Needham and-M; M. Kochhar, J. Chromatogr., iii .(1975) 422. 75 -A. P. De Leenheer,‘ S. S. Rigo, (3. F. 9elijktin.s and P. M:van Vaerenbergh, J. Chromatogr., 92 (1974) 339. 76 R. K. Gilpin, J. A. Kspi and C. A. Janicki, J. Chromzzfugr., -107 (197.5) 115.. 77 F. Bailey and P. N. Brittain, J. Chrumato&., 83 (1973) 431. 78 L. T. Grady and S. E. Hays, J. PhCmm_ Stii., 62 (1973) 456. 79 R E. Lovins and S. R. Eliis, Anal. Cbn~, 45 (1973) 1553. .80 M. Martin and G. Guiochon, Bull. Sac. Chim. Fr., (1973) 161. 81 J. S. Mayell and C. F. Hishey, Anal. Chem., 46 (1974) 449. 82 A. F. Michaelis and D. W. Comish, J_ Pirclrm. Sci., 62 (1973) 1399. 83l.S. G. Perry, Chem. EWr_, 7 (1971) 366. .84 T. M. Rajcsanyi and .L. Otvos, Separ_ Purif Metho& 2 (1973) 361. 85 R G. Muusze and J. F. K. Huber, J. Cirrotiiaiogr, Sci., 12 (1974) 779. 86 W- Lindner, R: W. Frei and W. Santi, J. Chromatogr.; 111. (1975) 365_ 87 J. F. Murray, Jr., G. R. Gordon, C. C. Gulledge and J. H; Peters, I. Chromatogr., 107 (1975) 67. 88~ W. Dunges and G. Naundorf, J. Chromarogr. Sci., I2 (i974) 655. 89 M. Roth.and A. Hunpai, J_ Chromatogr., 83 (1973) 353. 90 N. A. Parr& b. Chromatogr. Sci., 12 (1974) 753. 91 P. J. Twichett and A. C. Moeat, J. Chromazogr.. Ill (1975) 149. 92 R. W. Frei, ReS. Develop., 25 (E974) 32. 93 F. Bailey, P. N. Brittain and B. F. Williamson, J. Chromatigr., 109 (19%) 305. .-
