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__

bUMMARY .-

A svey of the literature h& been carried out, covering pharmaceutical ap-
plititioks of high-performance liquid chromatography (HPLC) from 19724975. The
&bstra&s are divided into sevea groups, vi,. alkaloids, antibiotics, nitrogen-
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 of
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 cbromatogra-
phy (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 im-
possible 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 slow-
ness_ _- .:.; -,.
.. 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
COrtkosterokis, ----,Betan~etbsone 17-v&r&e; --em--.bydroc&isone acetate; +pred-.
nisobne auztate; ------, txiardnolone acetonide;-, 3uocinobne wotide.
 .-__.. -.’

‘.ti@j&&S OF HPw IN PEIAEWkEtiCAL

--
iiZ&TR% 75
_.’- __ ._
. :-
.!@e Et&&& _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 utihsa-
tion 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 ap-
plicabibty from the analjks of plant extracts to the quantitative analysis of multi-
component 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 any-
thing about the chemica! purity of the medicinal. On the basis of these tests it is di%i-
cult 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 adsor-
bants, 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, &ethyl&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
 .. r.‘&.&y
yi- ._

. ..- .’

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-aqueous- the 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 metha-
nol, 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 con-
taining .dichloromethane, methanoi, and water on the. adsorbant. Zorbax-Sil. The
second, by Twitchettgl, ev&ates the use of octadecylsilane chemically bonded to. 10-
m 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 chro-
Satograph 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

t~~twb~~2?reasofpumpsand~et~tow.Pumpsya~~edinc~~pie~~ 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 uni-
versa1detector. 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

TABLE I
ULTRAYiOLET ABSORPTiON OF CHROMOPHORIC GROUPS IN STEROIDS

Chromophores Ubravfokt absorph~on

Carbonyl 170-200
280-300

Double bond 180-225 204 3,300

a$-Unsatura& ketone 230-270 253 11,200

Conjugated dimes (different

rings) 232 21,500
239 23,.5ocl
Con&ate4 dienes (same 248 16.000
ring) 262 7,700
a3.’ 271
282
11,400
I1*!300
293 6900

_Conjugated poiyenes 306 14,saO

284 =,aao

223 13,500
256 11,900
I5J#
~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~grarnof 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/&; at-
tenuation,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,sulpha-
metl@ne. Column,(20~~ x 4.6 mm LD.) packed&th S-pmLichrosorb SMO; solventsystem,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 = fi-
Apooxyt&acycEne;2 = suIpbamezatbine (internalstandard);3 = a-apooxytetracycline;4 = oxy-
tetracycline;
5 =a&&ooxyte&acycl&; 6 = unknown.

Fig_ 7 showkthe di&rence in sensitivityof the impurities in this mixture when moni-
tored at 254 n.m rather than 273 mn. F&. 8 demonstrates the usefullnessof a double-
b&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;
..

:
Ellal~
:. w&e

.&I
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&e-
form of data handling. This procedure has been carried out for -theanalysis of chlbr-
hexidine=. 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,
9 R. A. H eauxk, K. R. La&k, J. D. M&Neil and R. W. Frei, 6. Chromarogr., 77 (1973) 425.
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.
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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.
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53 A. X Butte&i&d and B_ A. Lo&e, JI Ckomorogr. Sc& 11 @m_ al.
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55 R A. Henry sd J. A.+S&nit, J. Chromafogr. Sci., 9 (1971) 513.
 _.
84 ..:<: .;:.‘-. .:-
.. .. 1i -1, : .: _; -.’ : -. ‘. _:_:;, ; .. ;: 1._y, _’ F-
y.__ :. :. ..>-._ B$i$Y
-:
I . . .
-% & 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
-58 S: L. Sarkaqtid Et V. Jogi, J. f?hr&~r~~__S&.; 12 (1974) 206. ..- .:I- ..- .-.-~:--. .:
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. ._
62 N. J,~Pound and. R_ .W. sears, C’ori;,J. Pkayt. Sci..,_S (1973) S+ : ._ .,...-. -. .I_
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.
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33 R.-W. Roes, J.Phnrm. Sci., 61 (1972) 1979.
59 F. Bailey and P.. N. Brittain, J. Pfzarm. Phar~aVc& 24 (1972) 425.. -.
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73 B. =-Penner, J’. Phkn. SC& 69 (1975) 1017.
74 L. L. Needham and-M; M. Kochhar, J. Chromatogr., iii .(1975) 422.
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(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_
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