Journal of Chromatography, 623 (1992) 3341 Elsevler Science Pubhshers B V , Amsterdam
CHROM
24 455
Efficient enantioselective separation and determination trace impurities in secondary amino acids (i.e., imino acids)
of
Janusz Zukowskl, Maria Pawlowska and Dame1 W Armstrong Department of Chemrstry, Unrversrty of Mwsourt-Rolla, RoNa, MO 65401 (USA)
(First received April
13th, 1992, revised manuscript
received June 19th, 1992)
ABSTRACT An R-( -)-1-(1-naphthyl)ethyl carbamoylated+cyclodextrm bonded phase m conJunctIon with a nonaqueous polar mobde phase was used for the highly selective enantloseparatlon of a number of secondary ammo acids after their pre-column derlvatlzatlon with 9-fluorenylmethyl chloroformate (FMOC) Under the condltlons employed, the FMOC reagent served to “lock” the ammo acid mto their existing conformation thereby preventmg the posslblhty of racemlzatlon Furthermore, It served to increase the sensltlvlty to the pomt that trace level enantiomerlc lmpurltles were easily detected Compared with separations that use traditional reversed-phase solvents, this method showed several advantages higher selectlvlty towards the ammo acid enantlomers investigated, shorter analysis times, faster equlhbratlon of the column, more stable baselme and more sensitive fluorescence detectlon The detection hmlts for FMOC derlvatlves of prolme, rrans-4-hydroxyprohne, crs-4-hydroxyprohne, pyroglutamlc acid, 3,4-dehydroprohne, thlaprohne, pemclllamme acetone adduct and plpecohc acid are m the low femtomole range The method was used for evaluation of enantloselectlvlty of a number of “optlcally pure” commercial ammo acid standards Enantlomerlc lmpuntles as low as 0 0001% (1 ppm) can be determined m of trace levels of D-ammo acids m the presence of large amounts of correspondmg (opposite) L some cases High precision determmatlon enantlomer at 1, 0 1, 0 01% and below are demonstrated
INTRODUCTION
Ammo acids form a large group of compounds of pharmaceutical and blochemlcal interest The stereolsomers of ammo acids differ m blologlcal activity, hence then- configuratlon and optical purity are very important m many fields of peptlde and polypeptlde chemistry Because D-ammo acids are rare m nature compared with the L enantlomers, their determmatlon m complex blologlcal samples reqmres selective, efficient and highly sensitive screening techniques As has been shown m recent years, the method of choice often 1s hlgh-perforCorrespondence to Dr D W Armstrong, Department of Chemistry, University of Mlssoun-Rolla, Rolla, MO 65401, USA
mance liquid chromatography (HPLC) with fluonmetnc detection In the past two decades, a number of different HPLC procedures have been developed for enantlomerlc separation of ammo acids using both direct and indirect methods The progress made m the field can be found m many original research papers and review articles and will not be discussed m detail here
[l-31 HPLC methods are often combined with pre- or post-column derlvatlzatlon m order to achieve hlghsensitivity fluorlmetrlc or photometric detection Many chromatographlc procedures use denvatlzatlon reagents such as 7-chloro-4-mtrobenzo-2oxa- 1,3-dlazole (NBD-Cl) [4-61, dansyl chloride (DNS-Cl) [7-l 21, phenyhsothlocyanate (PITC) [ 13151, ortho-phthalaldehyde-mercaptoethanol (OPA-
34
ME) [16-201, 9-fluorenylmethyl chloroformate (FMOC-Cl) [21-251 and naphthalenedlaldehyde and analogues [26,27] The above methods have been successfully used for chn-al and achu-al separation of a number of ammo acids However, each method has distinct advantages and disadvantages It 1s especially dlfficult to conduct the DNS-Cl derlvatlzatlon reaction adequately when the ammo acids are present m low concentration m complex mixtures [2X] The OPAME derlvatlzatlon 1s more rapid and sensltlve, but its use 1s limited to primary ammo acids The PITC reagent requires an evaporation of the sample to remove excess of reagent prior to HPLC In addltlon, all of these derivatives show only limited stab&y The labeling with FMOC-Cl avoids the problems mentioned above and combines high sensltlvlty m fluorescence detection with favorable chromatographlc properties and ease of denvatlzatlon for both primary and secondary ammo acids This paper presents the results of enantloseparatlon of a number of secondary ammo acids (ammo acids) including naturally occurring prohne, hydroxyprolmes, plpecohc acid and pyroglutamlc acid Their physlologlcal and pathological roles have received attention from numerous workers [29-311 Several different HPLC techniques have been used for quantitative achlral determination of secondary ammo acids m blologlcal samples [5,22, 25,32-381, however a precise, accurate and sensitive method for choral determination 1s still needed The enantlomerlc separation of the FMOC-ammo acids was obtained on a R-( -)-l-(-naphthyl)ethyl carbamoylated-fi-cyclodextrm (RN-P-CD) column which has been previously used for successful resolution of a large variety of enantlomers m the normalphase and reversed-phase mode [22,39,40] In the present study the RN-/?-CD column, operated with a nonaqueous polar mobile phase, was used for separation of secondary ammo acids after their pre-column derlvatlzatlon with FMOC-Cl To our knowledge, it 1s the first enantloseparatlon of highly fluorescent FMOC derivatives of prohne [22], plpecolic acid and their analogues The method was used for determination of enantlomerlc contammatlon of a number of “optically pure” commercial ammo acid standards
J Zukowskt
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623 (1992) 33-41
EXPERIMENTAL
Apparatus
The HPLC system consisted of three pumps (LC-6A, Shlmadzu, Kyoto, Japan), a system controller (SCL-6B, Shlmadzu), UV detector (SPD-6A, Shlmadzu), fluorescence detector (RF-535, Shlmadzu), swltchmg valve (Rheodyne, Cotatl, CA, USA) and 0 2-~1 injector valve (Valco, Houston, TX, USA) The columns were 100 x 4 6 mm C1s, 250 x 4 6 mm acetylated+cyclodextrm (AC-/?-CD) and RN-P-CD (Astec, Whlppany, NJ, USA) Chemicals
Ammo acids were purchased from different sources listed m Table II Acetomtnle, water, acetic acid and trlethylamme were of HPLC grade and supplied from EM Science (Glbbstown, NJ, USA) Procedure
Derlvatlzmg agent FMOC-Cl was purchased from Sigma (St Louis, MO, USA) Derlvatlzatlon was performed according to ref 21 D-Thiaprohne was obtained by racemlzatlon of L-thlaprohne m boiling 6 M HCl solution trans-4-Hydroxy-D-prohne was obtained by eplmerlzatlon of as-4-hydroxy-L-prolme according to ref 41 Isomerlzatlon degree of both racemlzatlon and eplmerlzatlon reactions was checked using a C,,/RN-P-CD column swltchmg method After derlvatlzatlon of post reaction mixtures with FMOC-Cl, 5 ~1 of the sample was injected onto the Cl8 column and chromatographed using acetomtrde-water-acetic acid (200 800 2, v/v/v) at 0 5 ml/mm A UV wavelength of 266 nm was used to monitor the effluent The switching valve was turned for 2 s after the signal reached the maximum of the standard retention time In this way a small portion of the elutmg peak of trans-4-hydroxyprohne or thlaprohne was introduced mto the chn-al column RESULTS
AND DISCUSSION
Optlmuatlon
of mobile phase composltron
Most FMOC-functlonahzed ammo acids can be resolved m either a conventional reversed-phase mode (hydro-organic solvents) or with a mobile phase conslstmg almost entirely of acetomtrlle (contammg small amount of glacial acetic acid and trlethylamme modifiers) The latter of these will be
J Zukowskl
et al / J Chromatogr
15k’
A
.
3s
623 (1992) 33-41 15,
B
a 14-r
L
IO-
%H,O
%H20
Fig 1 influence of water concentration m the mobile phase on (A) the retention and (B) enantloselectlvlty obtained on AC-B-CD (W) and RN-/?-CD (A) column for D, L-prohne Eluent acetomtnle-water-0 6% tnethylamme-0 4% acetic acid Flow-rate I ml/mm
referred to as the “polar organic mobile phase” As can be seen m Figs 1 and 2, for prohne, separations done with polar organic mobile phase are generally preferable to those done with a hydroorganic mobile phase This 1s because the enantloselectivity (a) 1s slgmlkantly greater with a polar organic mobile phase, while retention times are less Fig 1 also shows that the R-( -)-l-(l-naphthyl)-
D
(A)
0
;0
40 ml”
Fig 2 Enantloseparatlon of D, L-prohne obtamed on RN-/J-CD column m (A) nonaqueous system, eluent acetomtnle-tnethylamme-acetic acid (1000 6 4), (B) under optimal condltlons m aqueous system Eluent water-acetomtrk+tr~ethylamme-acetic acid (850 150 6 4) Flow-rate 1 ml/mm The time scale at the bottom of the figure applies to both chromatograms (A) and (B)
ethyl carbamoylated-,+cyclodextrm column 1s generally more selective for the FMOC-ammo acids than 1s the acetylated-fi-CD column It 1s also interesting to note that m the reversed-phase mode, the large increases m retention with mobile phases containing more than 50% buffer did not produce correspondmg improvements m the separation or the enantloselectlvlty (Fig 1) In the case of the acetylated-/-CD stationary phase, the addition of as little as 4% (v/v) water to the mobile phase negated the enantloselectlvlty (Fig 1B) These results indicate that the choral recogmtlon mechanism may be dependent on the mobile phase composltlon In the case of l-( l-naphthyl)ethyl carbamoylated-fl-cyclodextrm columns, this phenomenon has been previously reported and discussed m detail [12,39,40] The aforementioned conclusion 1s also supported by the results shown m Fig 3 The addition of water mto an acetomtrlle mobile phase influences not only the selectlvlty, but also efficiency of the column The plots shown m Fig 3 can give some mformatlon on the molecular recogmtlon mechanism Followmg the general nonequlhbrmm theory developed by Glddmgs [42], the overall plate height (H) for hqmd-solid chromatography (LSC) can be expressed as H=A+:+c$+c& Zone
spreading
(1) 1s due
to
three
mdependent
J Zukowskl
et al 1 J Chromatogr
623 (1992) 33-41
the mass-transfer effects controlled by diffusion m the mobile phase Since
%H20
Fig 3 Influence of water concentration m the mobde phase on efficiency of RN-/l-CD column, A = L-prohne, 0 = D-prohne Chromatographlc condltlons as m Fig 1
sources flow pattern effects (A), longltudmal dlffuslon (B) and mass transfer effects (C) Flow pattern effects depend on the structure of the porous support material and are independent of the eluent velocity (u) Ordinary molecular diffusion m the flow dn-ectlon contnbutes most at low velocltles and m LC the term B/u 1s small Mass transfer effects contribute most m high-speed runs In adsorptlon chromatography they are controlled by two baslcally different mechanisms or some combmatlon thereof dlffuaon-controlled sorption and desorptlon rates orlgmatmg m the mobile phase (C,) and adsorptlon-desorptlon kinetics (C,) For the same chromatographlc system, the contnbutton of the first three terms to H 1s the same for both enantlomers According to
cc24 k
kd
k (1 + ,1)2
where q 1s the geometrical parameter, kd IS the desorptlon rate and k’ 1s the capacity factor, the significant differences m column efficiency observed for prolme enantlomers m the non-aqueous system indicates that there 1s a considerable difference m adsorption-desorptlon kinetics for both enantlomers Indeed, for the stronger retarded L-enantlomer, the rate of the adsorptlon-desorptlon process 1s about 2 times lower than for the D enantlomer The change m the eluent composltlon influences
where w 1s a dlmenslonless constant, dp 1s the particle size and D, 1s the dlffuslon coefficient m the mobile phase The change m the eluent composltlon also influences the dlffuslon velocity because of changes m the solvent vlscoslty (D, z l/r, where q = vlscoslty) However, the dramatlc decrease of the height equivalent to a theoretical plate (HETP) values for both prohne enantlomers cannot be attributed to the mass transfer effects m the mobile phase The effect observed is Just opposite to that which should be expected from theory the addition of water induces a large increase m the solvent vlscoslty which should also increase the contrlbutlon of C, term of the plate height Moreover, the addition of water slgmficantly decreases capacity factors m the discussed region [O-4% (v/v) water] Because K/(1 + k1)2 (eqn 2) is an increasing function with decreasing k’ values (k’ > l), the reduction m the HETP values also cannot be due to changes m the capacity factors Despite a large increase m solvent viscosity [q (water) = 1 cP, 9 (acetonitrile) = 0 34 cP] m the true reversed-phase mode, the plate heights for both enantlomers are slmllar, and only slightly dependent on eluent composltlon m the range studied Interpretation of the data leads to the assumption that the addition of water to the eluent changes the choral recogmtlon mechamsm and influences the kinetic process of sorption-desorptlon m the stationary phase which results m narrower peaks The HETP values obtained m aqueous systems are lower (for the similar capacity factor as indicated m Fig 3) compared with HETP found m the nonaqueous system The results suggest the existence of at least two types of recogmtlon mechamsms which differ m the rate of adsorptlon-desorptlon process It should be mentioned that m contrast to tradltlonal aqueous systems, the recogmtlon mechanism m nonaqueous systems 1s still not clear It has been postulated that an mcluslon complexatlon may not be occurring m these conditions [43] Rather, a more external adsorption at the mouth of the cyclodextrm cavity could account for the observed separations
J Zukowskl
et al / J Chromatogr
623 (1992) 33-41
-1
A
Y .
31
a 1.5
15-
15 10-
14
5-
0-c 00
I
1
05
10
.
I
15
12
.
20
00
20
on total amount of trlethylamme and acetic acid dnd their relative ratlo Column RN-P-CD Eluent acetomtnle-tnethylamlne-acetlc
mcludmg the apparent size selectivity between CI,j3 and y-cyclodextrm [43] Fig 2 shows the enantloseparatlon of a prohne racemate obtained on RN-/&CD column m the nonaqueous mode and under optimal condltlons m a water-rich system The prehmmary mvestlgatlons have shown that the water-free system has several advantages over the aqueous one mcludmg greater resolution, faster equlhbratlon of the column, more stable base-line and lower detection hmlts Thus, the RN-P-CD column which exhibited high selectlvlty toward FMOC-D, L-prohne, and a nonaqueous eluent was chosen for the detailed study on the optlmlzatlon of enantloseparatlon of secondary ammo acids As indicated m Fig 4, retention and selectlvlty of nonaqueous systems can be effectively regulated by changes m two parameters of the mobile phase the total amount of trlethylamme and acetic acid added and their relative ratios Fig 4 presents the typical dependence for all ammo acids mvestlgated This behavior may be used for an optlmlzatlon of separation factors for any analogous separation problem Fig 5 shows the influence of tnethylamme and acetic acid concentration on the enantloseparatlon of plpecohc acid racemate of the method data for all racemlc
15
96 Trletbylam&v/v
56 Tnethylamme,v/v
Fig 4 Dependence of (A) the retention and (B) enantloselectlvlty added to neat acetomtnle Test compound D, L-prohne Trlethylamme acetlc acid W = 2 1, 0 = 2 3, A = 1 2
Selectwlty and sensltwlty The chromatographlc
10
05
mix-
tures investigated are summarized m Table I The selectivity of the system was regulated by adjustmg the mobile phase composltlon to achieve base-line resolution with a reasonable retention time The combmatlon of good selectlvlty (x 1 15-l 50) and high efficiency results m high resolution factors for FMOC prohne enantlomers and its analogues The enantloseparatlon of plpecohc acid was achieved with an eluent containing lower concentrations of tnethylamme and acetic acid, which resulted m a longer analysis time The separation of mpecotlc acid (an analogue of plpecohc acid) was not possible It should be mentioned that high selectlvltles and resolutions listed m Table I are more than adequate
0
10 0
15
1
25 min
Fig 5 Effect of total amount of trlethylamme and acetx acid per 1000 ml of acetomtrde m the moblle phase on the resolutlon of D, L-plpecohc acid obtained on RN-B-CD column Flow-rate 1 ml/mm Eluent (A) acetomtnle-0 6% tnethylamme0 9% acetlc acid, (B) acetomtnlti 3% tnethylamme-0 45% acetlc acid, (C) acetomtrlle-0 12% tnethylammti 18% acetic acid
38 TABLE
J Zukowskl
et al / J Chromatogr
623 (1992) 33-41
I
SEPARATION DATA FOR RACEMIC MIXTURES WITH A NONAQUEOUS MOBILE PHASE
Name
Structure
Prolme
CT ?
OF SECONDARY
AMINO
ACIDS ON RN-j&CD
Eluent”
k;
k;
a
6 4 1000
29
44
15
41
6 4 1000
13
96
13
23
6 4 1000 3 2 1000
18 32
22 41
12 13
17 21
6 4 1000
19
24
13
33
6 4 1000
26
36
14
35
6 4 1000
19
22
12
26
6 4 1000 3 2 1000
12 19
14 23
12 12
15 20
6 4 1000 3 2 1000 1 5 1 1000
22 35 62
23 38 69
11 11 11
11 14 17
COLUMN
OPERATED
coon
rrans-4-Hydroxyprohne
\,)
‘COOH I
cu-4-Hydroxyprolme
COOH
Pyroglutamlc
acldb COOH k
0
3,4-Dehydroprolme
N I li
COOH
Thlaprolme COOH
Pemcdlamme
Plpecohc
acid
acetone
adduct
a Tnethylamme-acetic acid-acetomtnle b Although this ts an amide It 1s known to react with a variety of acid chlorides as well ’ R, = Resolution
[49,50] In this work, we found that It reacts with FMOC-Cl
J Zukowskz
et al / J Chromatogr
39
623 (1992) 3341
t
(A)
0 hour
12 hours’
0% ll
1
4%D
24 hours 4
(B)
(Cl
’
48 hours 22% D
7%D
D
L.LlLL _u-.!
0
-
10 ml”
Fig 6 Change of enantlomerlc ratlo of thlaprohne durmg the racemlzatlon reactlon (see Expenmental) Eluent acetomtnletnethylamlne-acetlc acid (1000 6 4) Flow-rate 1 ml/mm
for determmatlons of racemlc mixtures However, as has recently been reported [44], the requirement for trace analysis are more rigorous Resolution factors greater than 2 for the racemlc mixture 1s an appropriate criterion for quantitative trace analysis applications The elutlon order was checked by the mjectlon of pure enantlomers or mixtures with different enantlomerlc ratios Smce for thlaprohne and tram-C hydroxyprohne, only L enantlomers were available, the enantlomers were prepared via racemlzatlon and eplmerlzatlon reactions, respectively, according to the procedure described m the Experimental section The degree of racemlzatlon was checked by HPLC Fig 6 shows the change of ratio of thlaprolme enantlomers during the racemlzatlon reaction The D enantiomer was eluted prior to the L enantlomer for all the FMOC-ammo acids exammed, which 1s an advantage because the L enantlomer 1s the dominant component m most blologlcal samples It has been shown that when traces are eluted before the maJor enantlomer the quantitative determination of choral composltlon becomes more accurate and precise [45,46], and trace detectability can be improved tremendously [47] The detection limits were calculated using a signal-to-noise ratio of 2 The detection limits for all enantlomers investigated are m the low femtomole range It was found that the sensltlvlty of the method depends on eluent composltlon The detection limit
0
20
0
20
0
20 ml"
Fig 7 Comparison of detectablhty of L-prohne m (A) aqueeluent [water-acetomtrde-trlethylammeacetlc acid ous (530 470 6 4)] and (B, C) nonaqueous eluent [acetomtnle-tnethylamme-acetlc acid (1000 6 4)] Column RN-P-CD Flowrate 1 ml/mm Injected amount (A) 7 4 fmol, (B) 7 4 fmol and (C) 1 4 fmol
for FMOC L-prohne, estimated m the nonaqueous system, was 5 times lower than was found for the system with a mobile phase contammg 53% of water (see Fig 7) This 1s probably due to the fact that for many aromatic and/or heteroaromatlc compounds water has a quenching effect on the fluorescence [48] In addition, as shown m Fig 7, the noise and baselme shifting 1s more prevalent m a water-nch system Practical applications This method was used for determination of enantlomerlc contammatlon m a number of commercial ammo acid standards The results are collected m Table II The chromatographlc condltlons are given m Table I Some level of enantlomerlc contammatlon was found m all commercial ammo acid samples As can be seen from the results m Table II the method enables trace and ultra-trace determination of optical purity for both L and D enantlomers Quantitative analysis of contaminating D enantlomers m L-ammo acids at 1, 0 1 and 0 01% gave very high precision (R S D for 4 measurements) of 0 3% (pyroglutamlc acid), 2 6% (czs-Chydroxyprohne) and 4 5% (FMOC-L-prohne), respectively For the L enantlomer, as the trace component, the situation 1s more complicated The problem assoclated with tailing from the enantlomerlcally rich component results m quantitative determmatlons with lower preclslon This 1s shown m Fig 8B The precision in quantitative analysis for optical purity of prolme enantlomers at the 0 1% level 1s 1 2% and
40
J Zukowskl et al 1 J Chromatogr
TABLE
623 (1992) 33-41
II
OPTICAL
PURITY
Chromatographlc
OF COMMERCIAL
condmons
SAMPLES
OF L- AND D-AMINO
ACIDS
as m Table I
Name
Source
Concentration of opposite enantlomer (%)
Eluent’
Standard devlatlond
D-Prohne L-Prolme
ICN Aldrich
07 05
A
0 08 0 006
FMOC-L-prolme
Fluka
0 02
A
0 0008
Irans-4-Hydroxy-L-proline*
Sigma
0 0 09b
A 0 008
cu-4-Hydroxy-D-prolme cu-4-Hydroxy-L-prohne
Sigma Sigma
04 15
B B
0 009 0 007
D-Pyroglutamlc L-Pyroglutamlc
Sigma Sigma
25 13
A A
0 04 0 004
Sigma
03
A
0 009
Sigma
03
B
001
3,4-Dehydro-L-prohne
Aldrich
04
A
004
L-Plpecohc
Aldrich
03
C
0 007
acid’ acid’
L-Thlaprolme L-Pemcdlamme
acetone
adduct
acid
Eluent acetomtnle-tnethylamme-acetic acid A = 1000 6 4, B = 1000 3 2, C = 1000 1 5 1 cu-4-Hydroxy-D-prolme, according to [43], trans-4-hydroxy+prolme epltienzes to crs-4-hydroxy-D-prohne Although this IS an amide it IS known to react with a variety ofacldchlorldes [49,50] In this work, we found that it reacts with FMOC-Cl as well n=5
-
(A)
L
!
(A)
(B)
(B)
I.
D
I
0
10
20
x_x I
0
10
20
TIME,MIN Fig 8 Chromatograms used to evaluate the enantlomerlc purity of (A) L-prohne (a puntied mixture) and (B) D-prohne (ICN) Eluent acetomtrde-tnethylammeacet~c acid (1000 6 4) Column RN-B-CD Flow-rate 1 ml/mm
0
30 0
: 30 “1”
Rg 9 Chromatograms used to evaluate the enantlon‘enc purity of (A) L-plpecohc acid (Aldrich) Eluent acetomtnle-tnethylamme-acetlc acid (1000 1 5 I), (B) crs-4-hydroxy+prohne (Slgma) Eluent acetomtrd~tr~ethylammc+acet~c acid (1000 3 2) The other condltlons as m Fig 8
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623 (1992) 33-41
11 1% for the D and L enantlomer, respectively The trace analysis precision for the L enantlomer found m this study was 1 8% (pyroglutamlc acid) at the 1% level and 11 1% (prohne) at the 0 1% level In conclusion, the present method gives high sensitivity and precision for the evaluation of trace levels of D ammo aads m the presence of large amounts of the correspondmg L enantlomer Fig 9 shows the chromatographlc separation of contamlnatmg D enantlomers in L-plpecohc acid and US-~hydroxy+prolme samples This technique can be easily adopted to routine analysis and has been used extensrvely m this laboratory to quantltate the D-prohne content m physlologlcal fluids down to the ppm level (Fig 8A) ACKNOWLEDGEMENT
Support of this work by the National Institute of General Medical Science (BMT lRO1 GM36292-04) 1s gratefully acknowledged REFERENCES 1 T J Ward and D W Armstrong, J Lzq Chromatogr, 9 (1986) 407 2 A M Krstulovlc (EdItor), Choral Separatzon by HPLC, Elhs Horwood, ChIchester, 1989 3 S AhuJa (EdItor), Chzral Separatzons by LC (ACS Symposzum Serzes, No 471), American Chemical Society, Washmgton, DC, 1991 4 R S Fager, G B Kutma and E W Abrahamson, Anal Bzochem, 53 (1973) 290 5 M Ahnoff, I Grundvlk, A Arfwldsson, J Fonsehus and B A Persson, Anal Chem , 53 (1981) 485 N Nlmura, A Toyama and T Kmoshlta, J Chromatogr , 234 (1982) 482 S Wemstem and S Werner, J Chromatogr , 303 (1984) 244 E Bayer, D Gram, B Katteneggar and R Uhnam, Anal Chem , 48 (1976) 1106 9 W Lmdner, I N le Page, G Davies, D E Seitz and B L Karger, J Chromatogr , 185 (1979) 323 10 K FuJimura, S Suzuki, K Hayashl and S Mosuda, Anal Chem , 62 (1990) 2198 11 S Allenmark and S Andersson, J Chromafogr , 351 (1986) 231 12 M L Hdton, S Chang, M P Gasper and D W Armstrong, J Lzq Chromatogr , (1992) m press 13 S A Cohen and D J Strydouz, Anal Bzochem ,174 (1988) 1 14 R L Hemrlkson and S C Meredith, Anal Bzochem, 136 (1986) 65 15 S Gunawan, N Y Walton and D M Freeman, J Chromatogr , 503 (1990) 177 16 M Roth, Anal Chem , 43 (1971) 880 17 E Gd, A Tlshbee and P E J Hare, J Am Chem Sot, 102 (1980) 5115 18 N Nlmura, T Suzuki, Y Kasahara and T Kmoshlta, Anal Chem, 53 (1991) 1380
41 19 1 Morettl, M Blranelh, C Cdrducci, A Pomecorvi and 1 Antonozq J Chromatogr, 511 (1990) 131 20 D W Armstrong, J D Duncan and S H Lee, Amzno Aczds, 1 (1991) 97 21 S Emarsson, B Josefsson and S Lagerkvlst, J Chromatogr, 382 (1983) 609 22 S Emarsson, J Chromatogr, 384 (1985) 213 23 S Emarsson, S Folestad, B Josefsson and S Lagerkvlst, Anal Chem , 58 (1986) 1638 24 M F Molnar and L A S Schroeder, J Chromatogr , 514 (1990) 227 D Fucks, J 0 Bosset and W Gmuer, 25 V Buetlkofer, Chromatographza, 31 (1991) 441 26 R G Caelson, K Srmlvasachar, R S Glvens and B K Matuszewskl, J Org Chem , 51 (1986) 3978 C M Riley, L A Sterson and J F 27 P De Montlgny, Stobaugh, J Pharm Bzomed Anal, 8 (1990) 419 28 D J Neadle and R J Polhtt, Bzochem J, 97 (1965) 607 29 S Lam and A Karger, J Lzq Chromatogr , 9 (1986) 291 30 A Master, Sczence (Washzngton, DC), 130 (1973) 33 31 E Glacobml, Y Namura and T Schmidt-Glenewmkel, Cell MO/ Bzol, 26 (1980) 135 and J P Borlel, J 32 G Bellon, A Malgras, R Rondoux Chromatogr , 278 (1983) 167 33 S Emarsson, B Josefsson, P Moeller and D Sanchez, Anal Chem, 59 (1987) 1191 34 L A Schdb, V D Flegel and D R Kmghton, J Lzq Chromatogr , 13 (1990) 57 35 H Nlshio and T Segawa, Anal Bzochem, 135 (1983) 312 36 Y Okano, M Kataoka, T Mlyata, H Monmoto, K Takahama, T Hltoshl, Y Kase, I Matsumoto and T Shmka, Anal Bzochem , 117 (1981) 196 37 E Bousgnet, G Romeo and L I Glannola, J Chromatogr , 344 (1985) 325 38 R Dlmahne and J R Reeve, Jr , J Chromatogr ,257 (1983) 355 39 A M Stalcup, S Chang and D W Armstrong, J Chromatogr, 540 (1991) 113 40 D W Armstrong, S -D Chang and S H Lee, J Chromatogr , 539 (1991) 83 41 D D Dzlewlatkowskl, V C Mascall and R I Rlolo, Anal Bzochem , 49 (1972) 550 42 J C Glddmgs, Dynamzcs of Chromatography, Dekker, New York, 1965 43 D W Armstrong, S Chen, C Chang and S Chang, J Lzq Chromatogr , 15 (1992) 545 44 T D Doyle, in S Ahqa (Editor), Chzral Separatzons by LC (ACS Symposzum Serzes, No 471), American Chemical Society, Washington, DC, 1991, p 27 45 E Busker, K Guenter and J Martens, J Chromatogr , 350 (1985) 179 46 K G Feltsma, B F H Drenth and R A de Zeeuw, J Chromatogr , 387 (1987) 447 47 J A Perry, J D Ratelke and T J Szczerba, J Chromatogr , 389 (1987) 57 48 H Lmgeman, W J M Underberg, A Takadate and A Hulshoff, J Lzq Chromatogr , 8 (1985) 789 49 K Imakl, H Nlwa, S Sakuyama, T Okada, M Toka and M Hayashl, Chem Pharm Bull, 29 (1981) 2699 50 K Imakl, S Sakuyama, T Okada, M Toda, M Hayasht, T Mlyamoto, A Kawasaki and T Okegawa, Chem Pharm Bull, 29 (1981) 2210
CHROM
24 455
Efficient enantioselective separation and determination trace impurities in secondary amino acids (i.e., imino acids)
of
Janusz Zukowskl, Maria Pawlowska and Dame1 W Armstrong Department of Chemrstry, Unrversrty of Mwsourt-Rolla, RoNa, MO 65401 (USA)
(First received April
13th, 1992, revised manuscript
received June 19th, 1992)
ABSTRACT An R-( -)-1-(1-naphthyl)ethyl carbamoylated+cyclodextrm bonded phase m conJunctIon with a nonaqueous polar mobde phase was used for the highly selective enantloseparatlon of a number of secondary ammo acids after their pre-column derlvatlzatlon with 9-fluorenylmethyl chloroformate (FMOC) Under the condltlons employed, the FMOC reagent served to “lock” the ammo acid mto their existing conformation thereby preventmg the posslblhty of racemlzatlon Furthermore, It served to increase the sensltlvlty to the pomt that trace level enantiomerlc lmpurltles were easily detected Compared with separations that use traditional reversed-phase solvents, this method showed several advantages higher selectlvlty towards the ammo acid enantlomers investigated, shorter analysis times, faster equlhbratlon of the column, more stable baselme and more sensitive fluorescence detectlon The detection hmlts for FMOC derlvatlves of prolme, rrans-4-hydroxyprohne, crs-4-hydroxyprohne, pyroglutamlc acid, 3,4-dehydroprohne, thlaprohne, pemclllamme acetone adduct and plpecohc acid are m the low femtomole range The method was used for evaluation of enantloselectlvlty of a number of “optlcally pure” commercial ammo acid standards Enantlomerlc lmpuntles as low as 0 0001% (1 ppm) can be determined m of trace levels of D-ammo acids m the presence of large amounts of correspondmg (opposite) L some cases High precision determmatlon enantlomer at 1, 0 1, 0 01% and below are demonstrated
INTRODUCTION
Ammo acids form a large group of compounds of pharmaceutical and blochemlcal interest The stereolsomers of ammo acids differ m blologlcal activity, hence then- configuratlon and optical purity are very important m many fields of peptlde and polypeptlde chemistry Because D-ammo acids are rare m nature compared with the L enantlomers, their determmatlon m complex blologlcal samples reqmres selective, efficient and highly sensitive screening techniques As has been shown m recent years, the method of choice often 1s hlgh-perforCorrespondence to Dr D W Armstrong, Department of Chemistry, University of Mlssoun-Rolla, Rolla, MO 65401, USA
mance liquid chromatography (HPLC) with fluonmetnc detection In the past two decades, a number of different HPLC procedures have been developed for enantlomerlc separation of ammo acids using both direct and indirect methods The progress made m the field can be found m many original research papers and review articles and will not be discussed m detail here
[l-31 HPLC methods are often combined with pre- or post-column derlvatlzatlon m order to achieve hlghsensitivity fluorlmetrlc or photometric detection Many chromatographlc procedures use denvatlzatlon reagents such as 7-chloro-4-mtrobenzo-2oxa- 1,3-dlazole (NBD-Cl) [4-61, dansyl chloride (DNS-Cl) [7-l 21, phenyhsothlocyanate (PITC) [ 13151, ortho-phthalaldehyde-mercaptoethanol (OPA-
34
ME) [16-201, 9-fluorenylmethyl chloroformate (FMOC-Cl) [21-251 and naphthalenedlaldehyde and analogues [26,27] The above methods have been successfully used for chn-al and achu-al separation of a number of ammo acids However, each method has distinct advantages and disadvantages It 1s especially dlfficult to conduct the DNS-Cl derlvatlzatlon reaction adequately when the ammo acids are present m low concentration m complex mixtures [2X] The OPAME derlvatlzatlon 1s more rapid and sensltlve, but its use 1s limited to primary ammo acids The PITC reagent requires an evaporation of the sample to remove excess of reagent prior to HPLC In addltlon, all of these derivatives show only limited stab&y The labeling with FMOC-Cl avoids the problems mentioned above and combines high sensltlvlty m fluorescence detection with favorable chromatographlc properties and ease of denvatlzatlon for both primary and secondary ammo acids This paper presents the results of enantloseparatlon of a number of secondary ammo acids (ammo acids) including naturally occurring prohne, hydroxyprolmes, plpecohc acid and pyroglutamlc acid Their physlologlcal and pathological roles have received attention from numerous workers [29-311 Several different HPLC techniques have been used for quantitative achlral determination of secondary ammo acids m blologlcal samples [5,22, 25,32-381, however a precise, accurate and sensitive method for choral determination 1s still needed The enantlomerlc separation of the FMOC-ammo acids was obtained on a R-( -)-l-(-naphthyl)ethyl carbamoylated-fi-cyclodextrm (RN-P-CD) column which has been previously used for successful resolution of a large variety of enantlomers m the normalphase and reversed-phase mode [22,39,40] In the present study the RN-/?-CD column, operated with a nonaqueous polar mobile phase, was used for separation of secondary ammo acids after their pre-column derlvatlzatlon with FMOC-Cl To our knowledge, it 1s the first enantloseparatlon of highly fluorescent FMOC derivatives of prohne [22], plpecolic acid and their analogues The method was used for determination of enantlomerlc contammatlon of a number of “optically pure” commercial ammo acid standards
J Zukowskt
et al / J Chromatogr
623 (1992) 33-41
EXPERIMENTAL
Apparatus
The HPLC system consisted of three pumps (LC-6A, Shlmadzu, Kyoto, Japan), a system controller (SCL-6B, Shlmadzu), UV detector (SPD-6A, Shlmadzu), fluorescence detector (RF-535, Shlmadzu), swltchmg valve (Rheodyne, Cotatl, CA, USA) and 0 2-~1 injector valve (Valco, Houston, TX, USA) The columns were 100 x 4 6 mm C1s, 250 x 4 6 mm acetylated+cyclodextrm (AC-/?-CD) and RN-P-CD (Astec, Whlppany, NJ, USA) Chemicals
Ammo acids were purchased from different sources listed m Table II Acetomtnle, water, acetic acid and trlethylamme were of HPLC grade and supplied from EM Science (Glbbstown, NJ, USA) Procedure
Derlvatlzmg agent FMOC-Cl was purchased from Sigma (St Louis, MO, USA) Derlvatlzatlon was performed according to ref 21 D-Thiaprohne was obtained by racemlzatlon of L-thlaprohne m boiling 6 M HCl solution trans-4-Hydroxy-D-prohne was obtained by eplmerlzatlon of as-4-hydroxy-L-prolme according to ref 41 Isomerlzatlon degree of both racemlzatlon and eplmerlzatlon reactions was checked using a C,,/RN-P-CD column swltchmg method After derlvatlzatlon of post reaction mixtures with FMOC-Cl, 5 ~1 of the sample was injected onto the Cl8 column and chromatographed using acetomtrde-water-acetic acid (200 800 2, v/v/v) at 0 5 ml/mm A UV wavelength of 266 nm was used to monitor the effluent The switching valve was turned for 2 s after the signal reached the maximum of the standard retention time In this way a small portion of the elutmg peak of trans-4-hydroxyprohne or thlaprohne was introduced mto the chn-al column RESULTS
AND DISCUSSION
Optlmuatlon
of mobile phase composltron
Most FMOC-functlonahzed ammo acids can be resolved m either a conventional reversed-phase mode (hydro-organic solvents) or with a mobile phase conslstmg almost entirely of acetomtrlle (contammg small amount of glacial acetic acid and trlethylamme modifiers) The latter of these will be
J Zukowskl
et al / J Chromatogr
15k’
A
.
3s
623 (1992) 33-41 15,
B
a 14-r
L
IO-
%H,O
%H20
Fig 1 influence of water concentration m the mobile phase on (A) the retention and (B) enantloselectlvlty obtained on AC-B-CD (W) and RN-/?-CD (A) column for D, L-prohne Eluent acetomtnle-water-0 6% tnethylamme-0 4% acetic acid Flow-rate I ml/mm
referred to as the “polar organic mobile phase” As can be seen m Figs 1 and 2, for prohne, separations done with polar organic mobile phase are generally preferable to those done with a hydroorganic mobile phase This 1s because the enantloselectivity (a) 1s slgmlkantly greater with a polar organic mobile phase, while retention times are less Fig 1 also shows that the R-( -)-l-(l-naphthyl)-
D
(A)
0
;0
40 ml”
Fig 2 Enantloseparatlon of D, L-prohne obtamed on RN-/J-CD column m (A) nonaqueous system, eluent acetomtnle-tnethylamme-acetic acid (1000 6 4), (B) under optimal condltlons m aqueous system Eluent water-acetomtrk+tr~ethylamme-acetic acid (850 150 6 4) Flow-rate 1 ml/mm The time scale at the bottom of the figure applies to both chromatograms (A) and (B)
ethyl carbamoylated-,+cyclodextrm column 1s generally more selective for the FMOC-ammo acids than 1s the acetylated-fi-CD column It 1s also interesting to note that m the reversed-phase mode, the large increases m retention with mobile phases containing more than 50% buffer did not produce correspondmg improvements m the separation or the enantloselectlvlty (Fig 1) In the case of the acetylated-/-CD stationary phase, the addition of as little as 4% (v/v) water to the mobile phase negated the enantloselectlvlty (Fig 1B) These results indicate that the choral recogmtlon mechanism may be dependent on the mobile phase composltlon In the case of l-( l-naphthyl)ethyl carbamoylated-fl-cyclodextrm columns, this phenomenon has been previously reported and discussed m detail [12,39,40] The aforementioned conclusion 1s also supported by the results shown m Fig 3 The addition of water mto an acetomtrlle mobile phase influences not only the selectlvlty, but also efficiency of the column The plots shown m Fig 3 can give some mformatlon on the molecular recogmtlon mechanism Followmg the general nonequlhbrmm theory developed by Glddmgs [42], the overall plate height (H) for hqmd-solid chromatography (LSC) can be expressed as H=A+:+c$+c& Zone
spreading
(1) 1s due
to
three
mdependent
J Zukowskl
et al 1 J Chromatogr
623 (1992) 33-41
the mass-transfer effects controlled by diffusion m the mobile phase Since
%H20
Fig 3 Influence of water concentration m the mobde phase on efficiency of RN-/l-CD column, A = L-prohne, 0 = D-prohne Chromatographlc condltlons as m Fig 1
sources flow pattern effects (A), longltudmal dlffuslon (B) and mass transfer effects (C) Flow pattern effects depend on the structure of the porous support material and are independent of the eluent velocity (u) Ordinary molecular diffusion m the flow dn-ectlon contnbutes most at low velocltles and m LC the term B/u 1s small Mass transfer effects contribute most m high-speed runs In adsorptlon chromatography they are controlled by two baslcally different mechanisms or some combmatlon thereof dlffuaon-controlled sorption and desorptlon rates orlgmatmg m the mobile phase (C,) and adsorptlon-desorptlon kinetics (C,) For the same chromatographlc system, the contnbutton of the first three terms to H 1s the same for both enantlomers According to
cc24 k
kd
k (1 + ,1)2
where q 1s the geometrical parameter, kd IS the desorptlon rate and k’ 1s the capacity factor, the significant differences m column efficiency observed for prolme enantlomers m the non-aqueous system indicates that there 1s a considerable difference m adsorption-desorptlon kinetics for both enantlomers Indeed, for the stronger retarded L-enantlomer, the rate of the adsorptlon-desorptlon process 1s about 2 times lower than for the D enantlomer The change m the eluent composltlon influences
where w 1s a dlmenslonless constant, dp 1s the particle size and D, 1s the dlffuslon coefficient m the mobile phase The change m the eluent composltlon also influences the dlffuslon velocity because of changes m the solvent vlscoslty (D, z l/r, where q = vlscoslty) However, the dramatlc decrease of the height equivalent to a theoretical plate (HETP) values for both prohne enantlomers cannot be attributed to the mass transfer effects m the mobile phase The effect observed is Just opposite to that which should be expected from theory the addition of water induces a large increase m the solvent vlscoslty which should also increase the contrlbutlon of C, term of the plate height Moreover, the addition of water slgmficantly decreases capacity factors m the discussed region [O-4% (v/v) water] Because K/(1 + k1)2 (eqn 2) is an increasing function with decreasing k’ values (k’ > l), the reduction m the HETP values also cannot be due to changes m the capacity factors Despite a large increase m solvent viscosity [q (water) = 1 cP, 9 (acetonitrile) = 0 34 cP] m the true reversed-phase mode, the plate heights for both enantlomers are slmllar, and only slightly dependent on eluent composltlon m the range studied Interpretation of the data leads to the assumption that the addition of water to the eluent changes the choral recogmtlon mechamsm and influences the kinetic process of sorption-desorptlon m the stationary phase which results m narrower peaks The HETP values obtained m aqueous systems are lower (for the similar capacity factor as indicated m Fig 3) compared with HETP found m the nonaqueous system The results suggest the existence of at least two types of recogmtlon mechamsms which differ m the rate of adsorptlon-desorptlon process It should be mentioned that m contrast to tradltlonal aqueous systems, the recogmtlon mechanism m nonaqueous systems 1s still not clear It has been postulated that an mcluslon complexatlon may not be occurring m these conditions [43] Rather, a more external adsorption at the mouth of the cyclodextrm cavity could account for the observed separations
J Zukowskl
et al / J Chromatogr
623 (1992) 33-41
-1
A
Y .
31
a 1.5
15-
15 10-
14
5-
0-c 00
I
1
05
10
.
I
15
12
.
20
00
20
on total amount of trlethylamme and acetic acid dnd their relative ratlo Column RN-P-CD Eluent acetomtnle-tnethylamlne-acetlc
mcludmg the apparent size selectivity between CI,j3 and y-cyclodextrm [43] Fig 2 shows the enantloseparatlon of a prohne racemate obtained on RN-/&CD column m the nonaqueous mode and under optimal condltlons m a water-rich system The prehmmary mvestlgatlons have shown that the water-free system has several advantages over the aqueous one mcludmg greater resolution, faster equlhbratlon of the column, more stable base-line and lower detection hmlts Thus, the RN-P-CD column which exhibited high selectlvlty toward FMOC-D, L-prohne, and a nonaqueous eluent was chosen for the detailed study on the optlmlzatlon of enantloseparatlon of secondary ammo acids As indicated m Fig 4, retention and selectlvlty of nonaqueous systems can be effectively regulated by changes m two parameters of the mobile phase the total amount of trlethylamme and acetic acid added and their relative ratios Fig 4 presents the typical dependence for all ammo acids mvestlgated This behavior may be used for an optlmlzatlon of separation factors for any analogous separation problem Fig 5 shows the influence of tnethylamme and acetic acid concentration on the enantloseparatlon of plpecohc acid racemate of the method data for all racemlc
15
96 Trletbylam&v/v
56 Tnethylamme,v/v
Fig 4 Dependence of (A) the retention and (B) enantloselectlvlty added to neat acetomtnle Test compound D, L-prohne Trlethylamme acetlc acid W = 2 1, 0 = 2 3, A = 1 2
Selectwlty and sensltwlty The chromatographlc
10
05
mix-
tures investigated are summarized m Table I The selectivity of the system was regulated by adjustmg the mobile phase composltlon to achieve base-line resolution with a reasonable retention time The combmatlon of good selectlvlty (x 1 15-l 50) and high efficiency results m high resolution factors for FMOC prohne enantlomers and its analogues The enantloseparatlon of plpecohc acid was achieved with an eluent containing lower concentrations of tnethylamme and acetic acid, which resulted m a longer analysis time The separation of mpecotlc acid (an analogue of plpecohc acid) was not possible It should be mentioned that high selectlvltles and resolutions listed m Table I are more than adequate
0
10 0
15
1
25 min
Fig 5 Effect of total amount of trlethylamme and acetx acid per 1000 ml of acetomtrde m the moblle phase on the resolutlon of D, L-plpecohc acid obtained on RN-B-CD column Flow-rate 1 ml/mm Eluent (A) acetomtnle-0 6% tnethylamme0 9% acetlc acid, (B) acetomtnlti 3% tnethylamme-0 45% acetlc acid, (C) acetomtrlle-0 12% tnethylammti 18% acetic acid
38 TABLE
J Zukowskl
et al / J Chromatogr
623 (1992) 33-41
I
SEPARATION DATA FOR RACEMIC MIXTURES WITH A NONAQUEOUS MOBILE PHASE
Name
Structure
Prolme
CT ?
OF SECONDARY
AMINO
ACIDS ON RN-j&CD
Eluent”
k;
k;
a
6 4 1000
29
44
15
41
6 4 1000
13
96
13
23
6 4 1000 3 2 1000
18 32
22 41
12 13
17 21
6 4 1000
19
24
13
33
6 4 1000
26
36
14
35
6 4 1000
19
22
12
26
6 4 1000 3 2 1000
12 19
14 23
12 12
15 20
6 4 1000 3 2 1000 1 5 1 1000
22 35 62
23 38 69
11 11 11
11 14 17
COLUMN
OPERATED
coon
rrans-4-Hydroxyprohne
\,)
‘COOH I
cu-4-Hydroxyprolme
COOH
Pyroglutamlc
acldb COOH k
0
3,4-Dehydroprolme
N I li
COOH
Thlaprolme COOH
Pemcdlamme
Plpecohc
acid
acetone
adduct
a Tnethylamme-acetic acid-acetomtnle b Although this ts an amide It 1s known to react with a variety of acid chlorides as well ’ R, = Resolution
[49,50] In this work, we found that It reacts with FMOC-Cl
J Zukowskz
et al / J Chromatogr
39
623 (1992) 3341
t
(A)
0 hour
12 hours’
0% ll
1
4%D
24 hours 4
(B)
(Cl
’
48 hours 22% D
7%D
D
L.LlLL _u-.!
0
-
10 ml”
Fig 6 Change of enantlomerlc ratlo of thlaprohne durmg the racemlzatlon reactlon (see Expenmental) Eluent acetomtnletnethylamlne-acetlc acid (1000 6 4) Flow-rate 1 ml/mm
for determmatlons of racemlc mixtures However, as has recently been reported [44], the requirement for trace analysis are more rigorous Resolution factors greater than 2 for the racemlc mixture 1s an appropriate criterion for quantitative trace analysis applications The elutlon order was checked by the mjectlon of pure enantlomers or mixtures with different enantlomerlc ratios Smce for thlaprohne and tram-C hydroxyprohne, only L enantlomers were available, the enantlomers were prepared via racemlzatlon and eplmerlzatlon reactions, respectively, according to the procedure described m the Experimental section The degree of racemlzatlon was checked by HPLC Fig 6 shows the change of ratio of thlaprolme enantlomers during the racemlzatlon reaction The D enantiomer was eluted prior to the L enantlomer for all the FMOC-ammo acids exammed, which 1s an advantage because the L enantlomer 1s the dominant component m most blologlcal samples It has been shown that when traces are eluted before the maJor enantlomer the quantitative determination of choral composltlon becomes more accurate and precise [45,46], and trace detectability can be improved tremendously [47] The detection limits were calculated using a signal-to-noise ratio of 2 The detection limits for all enantlomers investigated are m the low femtomole range It was found that the sensltlvlty of the method depends on eluent composltlon The detection limit
0
20
0
20
0
20 ml"
Fig 7 Comparison of detectablhty of L-prohne m (A) aqueeluent [water-acetomtrde-trlethylammeacetlc acid ous (530 470 6 4)] and (B, C) nonaqueous eluent [acetomtnle-tnethylamme-acetlc acid (1000 6 4)] Column RN-P-CD Flowrate 1 ml/mm Injected amount (A) 7 4 fmol, (B) 7 4 fmol and (C) 1 4 fmol
for FMOC L-prohne, estimated m the nonaqueous system, was 5 times lower than was found for the system with a mobile phase contammg 53% of water (see Fig 7) This 1s probably due to the fact that for many aromatic and/or heteroaromatlc compounds water has a quenching effect on the fluorescence [48] In addition, as shown m Fig 7, the noise and baselme shifting 1s more prevalent m a water-nch system Practical applications This method was used for determination of enantlomerlc contammatlon m a number of commercial ammo acid standards The results are collected m Table II The chromatographlc condltlons are given m Table I Some level of enantlomerlc contammatlon was found m all commercial ammo acid samples As can be seen from the results m Table II the method enables trace and ultra-trace determination of optical purity for both L and D enantlomers Quantitative analysis of contaminating D enantlomers m L-ammo acids at 1, 0 1 and 0 01% gave very high precision (R S D for 4 measurements) of 0 3% (pyroglutamlc acid), 2 6% (czs-Chydroxyprohne) and 4 5% (FMOC-L-prohne), respectively For the L enantlomer, as the trace component, the situation 1s more complicated The problem assoclated with tailing from the enantlomerlcally rich component results m quantitative determmatlons with lower preclslon This 1s shown m Fig 8B The precision in quantitative analysis for optical purity of prolme enantlomers at the 0 1% level 1s 1 2% and
40
J Zukowskl et al 1 J Chromatogr
TABLE
623 (1992) 33-41
II
OPTICAL
PURITY
Chromatographlc
OF COMMERCIAL
condmons
SAMPLES
OF L- AND D-AMINO
ACIDS
as m Table I
Name
Source
Concentration of opposite enantlomer (%)
Eluent’
Standard devlatlond
D-Prohne L-Prolme
ICN Aldrich
07 05
A
0 08 0 006
FMOC-L-prolme
Fluka
0 02
A
0 0008
Irans-4-Hydroxy-L-proline*
Sigma
0 0 09b
A 0 008
cu-4-Hydroxy-D-prolme cu-4-Hydroxy-L-prohne
Sigma Sigma
04 15
B B
0 009 0 007
D-Pyroglutamlc L-Pyroglutamlc
Sigma Sigma
25 13
A A
0 04 0 004
Sigma
03
A
0 009
Sigma
03
B
001
3,4-Dehydro-L-prohne
Aldrich
04
A
004
L-Plpecohc
Aldrich
03
C
0 007
acid’ acid’
L-Thlaprolme L-Pemcdlamme
acetone
adduct
acid
Eluent acetomtnle-tnethylamme-acetic acid A = 1000 6 4, B = 1000 3 2, C = 1000 1 5 1 cu-4-Hydroxy-D-prolme, according to [43], trans-4-hydroxy+prolme epltienzes to crs-4-hydroxy-D-prohne Although this IS an amide it IS known to react with a variety ofacldchlorldes [49,50] In this work, we found that it reacts with FMOC-Cl as well n=5
-
(A)
L
!
(A)
(B)
(B)
I.
D
I
0
10
20
x_x I
0
10
20
TIME,MIN Fig 8 Chromatograms used to evaluate the enantlomerlc purity of (A) L-prohne (a puntied mixture) and (B) D-prohne (ICN) Eluent acetomtrde-tnethylammeacet~c acid (1000 6 4) Column RN-B-CD Flow-rate 1 ml/mm
0
30 0
: 30 “1”
Rg 9 Chromatograms used to evaluate the enantlon‘enc purity of (A) L-plpecohc acid (Aldrich) Eluent acetomtnle-tnethylamme-acetlc acid (1000 1 5 I), (B) crs-4-hydroxy+prohne (Slgma) Eluent acetomtrd~tr~ethylammc+acet~c acid (1000 3 2) The other condltlons as m Fig 8
J Zukowskz
et al / J Chromatogr
623 (1992) 33-41
11 1% for the D and L enantlomer, respectively The trace analysis precision for the L enantlomer found m this study was 1 8% (pyroglutamlc acid) at the 1% level and 11 1% (prohne) at the 0 1% level In conclusion, the present method gives high sensitivity and precision for the evaluation of trace levels of D ammo aads m the presence of large amounts of the correspondmg L enantlomer Fig 9 shows the chromatographlc separation of contamlnatmg D enantlomers in L-plpecohc acid and US-~hydroxy+prolme samples This technique can be easily adopted to routine analysis and has been used extensrvely m this laboratory to quantltate the D-prohne content m physlologlcal fluids down to the ppm level (Fig 8A) ACKNOWLEDGEMENT
Support of this work by the National Institute of General Medical Science (BMT lRO1 GM36292-04) 1s gratefully acknowledged REFERENCES 1 T J Ward and D W Armstrong, J Lzq Chromatogr, 9 (1986) 407 2 A M Krstulovlc (EdItor), Choral Separatzon by HPLC, Elhs Horwood, ChIchester, 1989 3 S AhuJa (EdItor), Chzral Separatzons by LC (ACS Symposzum Serzes, No 471), American Chemical Society, Washmgton, DC, 1991 4 R S Fager, G B Kutma and E W Abrahamson, Anal Bzochem, 53 (1973) 290 5 M Ahnoff, I Grundvlk, A Arfwldsson, J Fonsehus and B A Persson, Anal Chem , 53 (1981) 485 N Nlmura, A Toyama and T Kmoshlta, J Chromatogr , 234 (1982) 482 S Wemstem and S Werner, J Chromatogr , 303 (1984) 244 E Bayer, D Gram, B Katteneggar and R Uhnam, Anal Chem , 48 (1976) 1106 9 W Lmdner, I N le Page, G Davies, D E Seitz and B L Karger, J Chromatogr , 185 (1979) 323 10 K FuJimura, S Suzuki, K Hayashl and S Mosuda, Anal Chem , 62 (1990) 2198 11 S Allenmark and S Andersson, J Chromafogr , 351 (1986) 231 12 M L Hdton, S Chang, M P Gasper and D W Armstrong, J Lzq Chromatogr , (1992) m press 13 S A Cohen and D J Strydouz, Anal Bzochem ,174 (1988) 1 14 R L Hemrlkson and S C Meredith, Anal Bzochem, 136 (1986) 65 15 S Gunawan, N Y Walton and D M Freeman, J Chromatogr , 503 (1990) 177 16 M Roth, Anal Chem , 43 (1971) 880 17 E Gd, A Tlshbee and P E J Hare, J Am Chem Sot, 102 (1980) 5115 18 N Nlmura, T Suzuki, Y Kasahara and T Kmoshlta, Anal Chem, 53 (1991) 1380
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