Anal. Chem. 1992, 64, 371-379 (5) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. J . Chem. Soc., Chem. Commun. 1981, 325-327. (6) Martin, S. A.; Biemann, K. Int. J. Mass Spectrom. Ion Processes 1987, 7 8 , 213-228. (7) Kenttamaa. H. I.; Cooks, R. G. Int. J . Mass Spectrom. Ion Processes 1985, 64, 79-83. (8) Wysockl, V.; Kenttamaa, H.; Cooks, R. G. Inf. J . Mass Spectrom. Ion Processes 1987. 75, 181-208. (9) Mabud. Md. A.; DeKrey, M. J.; Cooks, R. G. Int. J . Mass Spectrom. Ion Processes 1985, 67, 285-294. (10) Dawson, P. H.; Douglas, D. J. I n Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley 8 Sons: New York, 1983; Chapter 6. (11) DeKrey, M. J.; Kenttamaa, H. 1.; Wysocki. V. H.; Cooks, R. G. Org. Mass Spectrom. 1988, 21, 193-195. (12) McLuckey, S. A.; Cooks, R. G. I n Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wlley 8 Sons: New York, 1983; Chapter 15. (13) Cooks, R. G.; Ast, T.; Mabud, Md. A. Int. J. Mass Spectrom. Ion Processes 1990, 100, 209-265. (14) Bier, M. E.; Schwartz, J. C.; Schey, K. L.; Cooks, R. G. Int. J . Mass Spectrom. Ion Processes 1990, 103, 1-19. (15) Willims, E. R.; Henry, K. D.; McLafferty, F. W.; Shabanowltz, J.; Hunt, D. F. J. Am. SOC.Mass Spectrom. 1990, 1 , 413-416. (16) LeMeillour. S.; Cole, R. B.; Clalret, F.; Fournler, F.; Tabet, J.-C.; Blasco, T.; Beaugrand, C.; Devant, G. Advances in Mass Spectrometry;
97 1
Heyden and Son: Chlchester, U.K., 1969; Vol. 11A, pp 236-237. (17) Roepstorff, P.;Fohlman, J. Btomed. Mass Spectrom. 1984, 7 1 , 601. (18) Barber, M.; Bordoli, R. S.; Garner, G. V.; Gordon, D. B.; Sedgwlck, R. D.; Tetler, L. W.; Tyler, A. N. Blochem. J. 1981, 197, 401-404. (19) Katakuse, I.; Desiderlo. D. M. Int. J. Mass S ~ e c t r mIon . Processes 1983, 54, 1-15. (20) Gaskell, S. J.; Rellly, M. H.; Porter, C. J. RapM Commun. Mass S m trom. 1988. 2 , 142-145. (21) Aberth, W. Anal. Chem. 1990. 62, 609-611. (22) Baeten, W.; Claereboudt, J.; Van den Hewel, H.; Claeys, M. Bbmed. Environ. Mass Spectrom. 1989, 18, 727-732. (23) Alexander. A. J.; Boyd, R. K. Int. J. Mass Spectrom. Ion Processes 1989, 90, 211-240. (24) Bolt, G.; w e n . S.; Leary, J. A. RapM Commun. Mass Spectrom. IBBO. .- .. , 4. ., 341-344. - . . - . ..
(25) Tabet, J.4.; et al. Unpublished resub. (26) Kelner, L.; Markey, S. P. Int. J . Mass Spectrom. Ion Processes 1984. 59. 157-167. (27) Williams, D. H.; Naylor, S. J. Chem. SOC.,Chem. Commun. 1987, 1406- 1409.
RECEIVED for review June 19,1991. Accepted November 18, 1991.
Determination of Phospholipids from Pulmonary Surfactant Using an On-Line Coupled Silica/Reversed-Phase High-Performance Liquid Chromatography System Laurent Michel Bonanno,' Benoit Andre Denhot,$Pierre Cyril Tchoreloff,$ Frangis Puisieux,I and Philippe Jean Cardot*,+ Laboratoire de Chimie Analytique et d'Electrochimie Organiques and Laboratoire de Biopharmacie et Pharmacotechnie, Centre Pharmaceutique de ChBtenay-Malabry, 5 avenue Jean-Baptiste ClBment, F92296 Chatenay-Malabry Cedex, France
A basic normal-phase HPLC separation of phospholipids can be improved by introducing a limited contribution of solvophobic retention. For this purpose, the effect of an additional aikyisiiica (C18) column of variable length coupled in series with a silica column was investigated. With increasing percentage of reversed phase in this system, the retention of phosphatidyigiyceroi increased. Phosphatidylinositol and phosphatidylserine were separated into molecular species. The "selective retention" defined in this study permits an evaiuatbn of the sohrophobk retention of phospholipids in the coupled system. An alternative cdumn switching procedure is used for specific applications of the biphasic separation on chosen phospholipids. With this system, determination of phosphatklylglycerol and six other phospholipids from pulmonary surfactant could be performed.
INTRODUCTION Neonatal respiratory distress syndrome (RDS) results from surfactant deficiency in the pulmonary alveoli of premature newborns. This disease leads to severe cellular lesions and high mortality for the most The surfactant is a complex mixture of phospholipids, neutral lipids, and proteins. Preliminary studies have demonstrated the quantitative and qualitative importance of phospholipids in the compo-
* Corresponding author.
t Laboratoire de Chimie Analytique et d'Electrochimie Organiques. Laboratoire de Biopharmacie e t Pharmacotechnie.
*
sition and in the biophysical properties of the surfactant."1° HPLC methods have been developed for the separation of phospholipid c l a ~ s e s ,and ~ ~ -some ~ ~ have been adapted to the analysis of pulmonary These methods use a mobile-phase gradient elution on normal-phase columns. In these cases the separations were just efficient enough for the qualitative and quantitative determination of some phospholipid classes. However, the resolution of some adjacent peak couples was too low for a quantitative evaluation especially in the case of dioleoylphosphatidylglycerol (Pg).20923v24 An HPLC method is proposed to analyze phospholipid classes in an isocratic mode. This method uses a coupled stationary-phase system combining silica and alkylsilica (Cl& gel columns. Competition in the elution process between a solvophobic mechanism (reversed-phase elution mode) and a hydrophilic contribution from the silica gel column leads to a resolution of the dioleoylphosphatidylglycerol (Pg) from the solvent front better than in the case of one single silica column. With the mixed phase, and of a column-switching device, the detection limit of dioleoylphosphatidylglycerol(Pg) can be as low as 0.1 pg, of other phospholipids 0.01 mg/mL. THEORY Phospholipids and Chromatography. Phospholipids are typically amphiphilic compounds. They can be classed according to their polar head and fatty acid chains (Figure 1). This duality can be utilized both in normal and reversed-phase chromatography. For elution under normal-phase conditions, the selectivity of the retention is based on the adsorption of the polar head to the silanol sites of the stationary phase. It permits the identification of phospholipid classes in tissue
0003-2700/92/0364-0371$03.00/00 1992 American Chemical Society
372
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
1 GLYCEROL POLAR HEAD
HYDROPHOBIC TAIL
PHOSPHOLIPIDS CLASSES
(X)= --CH, --CHOH-- C% OH Phosphatidylglycerul --CH, --CH--NH, I
Phosphatidylserine
COOH
c h
Phosphatidylinositol
3
/
- - ( C H z ) , - - N ( C S :) --(CH,),
--NH3
Phosphatidylinositol Phosphatidylethanolamine
(RZ) Lysed: Lysophosphatidyl. MOLECULAR SPECIES
P-P
( R I ) AND (Ill) iLateral fatty acid ehsiw Sslumted or umalumled ex~mplrr:( R l = U = oleic arid): Dioleylphospkslidyl. ( R I = R 2 = n-kxadrcmole acid): Dipslmito,lphorphitidyl.
Figure 1. Amphiphilic nature of phospholipids.
e~tracts.ll-'~The reversed-phase solvophobic retention mechanism of some dioleoylphosphatidylcholines (Pc) has been demonstrated with mobile phases at low concentrations of water (5%).25-27 In these cases, the retention properties of phosphatidylcholines depends on the structure of their aliphatic chains (number of mbons, number of double bonds). To date, these separations have been performed using two chromatographic systems independently: normal phase to identify the classes, and reversed phase to identify the individual species. The coupling of these phases can improve the separation of both the classes and the species in a single analysis, according to the fact that in a given mobile phase, some phospholipids can have a predominant adsorption retention mechanism and others a predominant solvophobic one. To demonstrate these possible mechanisms, three chromatographic systems have been set up, as described in the experimental section (Figure 2). In the first one called "reference column", a single normal-phase column is used (Figure 2.1). The second system is a serial connection of the reference column with normal (Si/Si coupling) or reversed (Si/Cls coupling) stationary phase column segments (Figure 2.2). A third system was set up consisting of a switching device which connects optionally the reference column to a series of reversed-phase columns (Figure 2.3). This system can direct the eluent to the reversed phase (Figure 2.4) or to an independent detection system (Figure 2.5). Characterization of Chromatographic Parameters in the Mixed-Phase System. For the first system, described as system I in the Experimental Section, a single silica column served as separator. Its void volume (uOsi) is taken as the reference for the comparison of retention values. The retention of the phospholipids in this system will be characterized by the classical capacity factor k '. In the second and third systems, a set of short columns coupled with the reference normal-phase column modifies the global void volume (u0.J of the separator. Its variation can be expressed by the following relation: nc
uo, = uosi
+ iCUO,~,(i) =l
PUMPB
LV B
PL'MPB
UVB
i?QsmQM ILpuI1ppi2 Figure 2. Line diagram of the HPLC apparatus used for the comparative selectlvlty studies of phospholipids in the three systems. (1) System I consists of a single analytical silica column defined as a "reference" column, Spherisorb 5-pm silica gel, (Brownlee, 220 mm X 4.6 mm), and a Brownlee cartriige holder. (2) System I1 consists of a connection in series betwen the reference silica column and several small alkylsllica (C18)columns (Spherlsorb 5-pm C18, 15 mm X 4.6 mm cartridges). (3) System 111 consists of a column-switching system with a sixport valve between the reference silica column and small alkyisillca (C18)columns (Spherisorb 5-pm CI8SFCC 15 mm X 4.6 cm cartridges). Schematic representation of the eluent stream In a six-port valve connected with a C18short column: (4) position 1 = on-line position between silica gel and alkylsilica (C18);(5)position 2 = independent elution and detection for each column after switching.
where nc is the number of columns (1 < nc < 7) and uOClcis the void volume of each additional column (silica or reversed phase). All the columns are set up in a single coupling holder, so the connection volumes can be assumed to be negligible. Figure 3 shows the linear increase of the void volume with the number of additional silica or reversed-phase columns (Results). Then eq 1 can be expressed as follows: u0, = uOsi
+ ncuOclc
(2)
The void volume of the short column can be expressed as a fraction of the reference system void volume: U0,lc = auosj (3) and the global void volume as defined in eqs 1and 2 can be expressed as u0, = uOsi(l + anc) (4) where a is the ratio of the void volume of the short column to the void volume of the reference column (a < 1). The retention volume of a solute in the biphasic system is the sum of the retention volume in the reference column urSi and the retention volume in the additional columns urclc. The
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
3.16
-
3
326
-
B 4
3.06
-
2.86
.
1e
2.66
4,
373
a.nc
Flgure 4. Theoretical variation of the logarlthm of the selective retention (SR) with the ratio of stationary phase B and number of short
columns nc. Dashed line: SR =
number of columns
m e 3. oependence of the total dead volume on the number of short silica or alkylsilica columns connected in series wtth a nc: (0) Si; ( 0 ) C18.
global capacity factor k', of a solute in that biphasic system can be expressed by ita capacity fador in the reference column k 'si and in the additional column system k 'clc: k', =
k'si
+ (anc)kL1, 1 + anc
(5)
This relation shows that at a given number and geometry of columns the global capacity factor can be calculated from the individual ones for each column type. Mixed System: Definition of the "SelectiveRetention". FloydB has demonstrated the relation between the selectivity and the adsorption energies in a system that contains two different kinds of retention mechanism, as encountered in a heterogeneous phase.29 In our case: a silica and alkylsilica gel mixed stationary phase system, the adsorption on the silanol sites represents one family of interactions and the other is related to the solvophobic interaction with the alkylsilica portion of the system. Different percentage of stationary phase volumes will produce different selectivities. A parameter called the "selective retention" (SR) is defiied to evaluate the retention of solutes under such conditions and is expressed by
SR = k',/k'si
(6)
klSi is the capacity factor of a solute in the silica gel column, and k'p the capacity factor of the same solute in the mixed system (silica column coupled with alkylsilica columnssystem 11). With the expression of k', (eq 5), SR can be described as follows: 1 + anc(k',lc/kki) SR = (7) 1 anc
+
When silica columns of the same type are coupled to the reference column, k & is equal to k 'si, and SR = 1independent of the number of columns. With a mixed coupled system of silica and alkylsilica, SR depends essentially on the k' ratio anck b18/k'si.With the knowledge of the retention properties of a compound in both systems (k'c,, and kki), it is then possible to calculate SR as a function of the proportion anc as shown in Figure 4 for a k'ratio varying from 0.01 (solute predominantly retained by adsorption) to 100 (solute elution under a solvophobic mechanism) and the variation of anc corresponding to the values of nc between 1and 7. The ratio of the void volumes was taken from the slope calculated in
1,
k'ratio between 0.01 and 100.
Figure 3. In Figure 4 we can distinguish two zones. For compounds mainly retained by an adsorption mechanism, SR is smaller than 1 (k'c,, < h i i ) : the global k'decreases as Cle columns are added to the system. This "adsorption zone" is quite restricted because the system is dominated by the silica column and adding the short C18columns essentially "dilutes" the effect of the silica column. For k > k 'si, SR is greater than 1: the global k'increases with each additional C18 column. The retention of a given component can be predicted and optimized by an accurate choice of the stationary-phase ratio. In the case of phospholipids, because of their amphiphilic nature, it will be possible for a given mobile phase to determine the contribution t o the global retention of their polar head (adsoption) and of their fatty acid chains (alkylsilica).
EXPERIMENTAL SECTION Standards and Reagents. The phospholipid standards (dioleoylphosphatidylglycerol = Pg; phosphatidylinositol = Pi; dioleoylphosphatidylethanolamine= Pe; phosphatidylserine = Ps; dioleoylphosphatidylcholine = Pc; oleoyllysophosphatidylcholine = LPC; sphingomyelin = SM) were purchased from Sigma. A standard mixture of phospholipids is prepared at 0.1 mg/mL in chloroform/methanol(2/1 v/v). Chloroform, methanol, and phosphoric acid where of Normapur quality (Prolabo); HPLC grade acetonitrile was from Carlo Erba. Natural Lung Surfactant Extraction. Natural surfactant is extracted from adult ox lung. The organ is sliced and washed in a saline solution (NaC10.9%). The liquid obtained is then centrifuged at 4900 m s - ~(4 "C, 5 min) to eliminate cellular fragments and a second centrifugation (360000 m s - ~ ,4 "C, 40 min) is performed. The precipitated surfadant is then suspended in triply distilled water. Finally, phospholipids and lipophilic proteins are extracted by a chloroform/methanol (2/1 v/v) mixture.30 After evaporation under Nz, dry samples are stored at 4 "C. The quality of the extract was controlled by biological methods.' Procedure. A Waters 6000A HPLC pump and a Rheodyne 7015 valve with a 20-pL loop were used. The detection was performed at 206 nm with a Shimadzu SPD2A spectrophotometer. Quantitation was carried out by integration of the peak areas by a Shimadzu CR3A integrator. The chromatographicmobile phase is a mixture of acetonitrile/methanol/phosphoricacid (99/3/1 v/v). The system is set up at 3 mL/min, with the column and solvent thermostated at 28 "C. I. Normal Phase (System I), Reference System. The column used is a Spherisorb 5-pm silica gel cartridge (22 cm X 0.46 cm) in a Brownlee holder (Figure 2.1). This chromatographic system will be referred to as the reference system in the following section. 11. Bidimensional Chromatography. Connection in Series (System II). Cartridges of Spherisorb 5-pm Cls, 1.5 cm X 0.46 cm (Soci6t6 Franpaise Chromato Colonne (SFCC),France) were
374
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992 F
Table I. Retention Characteristics of Synthetic Phospholipids in System I with One Single Silica Column
L'
0.008 au
phospholipids
k 'si
Ne,
Pi Ps Pe Pc LPC
0.5
nd 1363 3632 5079 3595 3280
2.1
3.7 7.1 13.4 14.9
SM
Pi
0.96
I
2
3
4
6
7
number of silica columns
Dependence of the selective retention versus the number nc of short silica column segments: (A)Ps = phosphatldylserlne; (B) Pe = dloleoylphosphatidylethanolamlne; (A) Pc = dioleoylphosphatldylchollne;(0)LPC = oleoyllysophosphatldylchollne. Figure 6.
I
0
I
2
I
4
I
6
I
a
I
10
I
12
i
14
Time (min) Figure 5. HPLC chromatogram of phospholipid standards on the reference silica column. Conditions: Spherisorb 5-pm silica (22cm X 0.46 cm); mobile phase acetonRrile/methanol/phosphoric acid (99/3/1 v/v); flow rate 3 mL/min; injection 20 I.LL of the standard. Mixture (0.1 mg/mL of each component): Pi = phosphatidylinositol; Pg = dloleoylphosphatidylglycerol; Ps = phosphatidylserine; Pe = dioleoylphosphatidylethanolamine; Pc = dioleoylphosphatidylcholine; LPC = oleoyllysophosphatidylcholine in chloroform/methanol (211). Detection is at 206 nm. Peaks: SF = solvent front containing Pg; Pi, Pser, Pe, Pc, LPC.
fitted directly after the reference silica column in the same Brownlee coupling holder (Figure 2.2). The alkylsilica (C18)columns could be replaced by catridges of Spherisorb 5-pm silica gel, 1.5 cm X 0.46 cm (SFCC). This chromatographic system will be called a Si/% coupling system in the following section. Column Switching (System ZZI). A six-port HPLC valve is interfaced with the reference system and the alkylsilica (C18) column (Figure 2.3). The valve can altematively connect the outlet of the silica column to: (1)the alkylsilica (CpJ columns (Figure 2.4) and (2) the UV detector A (Figure 2.5). At injection the valve is set to position 1;the eluate from the silica column passes through the alkylsilica columns and the UV detector A. A total of 1min 40 s after the injection, after the entire solvent front has been transferred to the reversed-phase column, the valve is switched to position 2. Thus the entire solvent front is transferred to the alkylsilica column, and the more retained eluate of the silica column is directed directly to the UV detector B. At this time, a second HPLC pump (B) supplies the alkylsilica column by port 6 (Figure 2). Both pumps use the same mobile phase acetonitrile/methanol/phosphoric acid (991311 v/v).
RESULTS Normal-Phase Mode. System I. A mixture of phospholipid standards (composition described in the legend of
Figure 5 ) was injected into the first system. The chromatogram in Figure 5 shows the separation of phospholipid classes:
phosphatidylserine (Ps),dioleoylphosphatidylethanolamine (Pe), dioleoylphosphatidylcholine (Pc), oleoyllysophosphatidylcholine (LPC). Dioleoylphosphatidylglycerol(Pg) is eluted in the solvent front and not specifically detected. The peak of phosphatidylinositol (Pi) is detected but quantification is made difficult because of its low resolution with the solvent front. The capacity factor (k'si) and plate height number of all the solutes and of sphingomyelin (SM) (not shown in the chromatogram) is reported in Table I. These values &Isi) will serve to calculate SR (eq 6) for each phospholipid in the biphasic system 11. Chromatography i n a Mixed-Phase System. I. Connection in Series (System 11). This system combines the reference silica column with a number of silica gel (Si/Si system) or alkylsilica (Si/CI8 system) columns. Void Volume of the Chromatographic System. The global void volumes of system I1 are calculated from the retention time of the mobile-phase peak. The graph in Figure 3 shows the linear relation between the void volume and the number of short silica and alkylsilica columns coupled with the reference silica column. The slope is 0.13 for silica gel columns and 0.14 for alkylsilica columns. This difference between the curves indicates a reproducible difference in packing density of the two types of columns. SilicalSilica Coupling (SilSi System In. The standard mixture of phospholipids was injected in system I1 equipped with one to seven silica columns. The selective retention of five phospholipids was calculated according to eq 6 and is reported in Figure 6. As in system I, the peak of phosphatidylglycerolwas not detected. The SR of other phospholipids scatter around 1, as predicted in the theory section.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992 971
“I
SF
9 0
0 0
n
1
2
3
4
5
6
number of reverse phase columns (ne)
Figure 8. Dependence of the capacity factor of Pg on the number nc of short C18 column segments. The capacity factor at nc = 0 is k’, = 0, 18. 3,5
I
0
I
I
4
I
I
0
I
I
12
Time (min ) Flgure 7. Separation of phospholipMs in system 11. Conditions: connection in a series of cartridges of Spherisorb 5-pm silica (22 cm X 0.46 cm) and three short alkylsilica (&) Spherisorb 5-pm CI8 (15 mm X 4.6 mm) columns; injection 20 pL of a mixture containing 0.25 mg/mL Pg = dkleoylphosphaWVlglycerd,Ps = phosphatldylserine, Pe = dideoylphosphatidylethahatidylethandamine,Pc = dioleoylphosphatidylline in chloroform/methanol (2/ 1 v/v); mobile phase acetonitrlie/methanol/phosphorlc acM (99/3/1; v/v/v); flow rate 3 mL/min. Peaks: SF = Went front;Pg, Ps. Note the fourth peak of Ps molecular specks.
2.5
v) p!
1,s
C
Table 11. Capacity Factor for Each Phospholipid (Psi Experimental Determination and khlsTheoretical Determination) phospholipids
pg
Pe Ps Pc LPC
k ’si
k’cl*
0.18 3.7 2.1 7.1 13.4
17.3 10.2 6.4 4.6 0
SilicalAlkylsilica Coupling (Si/C18System II). Elutions of the same mixture were carried out in biphasic systems containing the same reference silica column coupled to one to seven alkylsilica columns. Figure 7 presents the chromatogram obtained in the case of three additional alkylsilica columns. In this system, the peak of dioleoylphosphatidylglycerol (Pg) is more retained and well resolved from the solvent front. With an increase of the alkylsilica proportion in the mixed-phase system, the retention of dioleoylphosphatidylglycerol increases linearly. The plot of the capacity factor k ; versus nc allows us to extrapolate the value of kki for nc = 0, which was found to be kki = 0.18, as shown in Figure 8. In order to determine the solvophobic or adsorption elution mechanism of the phospholipids, the selective retentions (SR) are calculated using both the k ’in the silica column (k’Si) and in the coupled system (k;) for different phospholipids. Figure
03
-0,s
a.nc Figure 9. Dependence of the selective retention on the^ ratlo of statlonery phase “a”and the number of short Cle columns “nc”: ( 0 )Pg = d k l e o y l p ; (A)Ps = 7 (W) ; Pe = dkleaylphosphatkfylethendamine; (A)Pc = (Ikleolylphosphatidy: (0)LPC = deoyltysophosphaUdylcholine; Theoretical values for each phospholipid are In dashed lines. Two zones appear: an adsorption (Pg, Ps, and Pe) and sohrophobic zone (Pc, LPC).
378
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
A
r
L I
I
I
I
n
Time ( min)
IC)
I
I
r
Pc Pi3
I
2
I
0.008 au
I-
IPI
i , I
I
i
’ser
I 0
I
i
0.008 au
I-
I
4
Time (min) Flgure 10. Molecular species of phosphatidylinositol in system 11. Conditlons: connection in series of cartrldges Spherisorb 5-pm silica (22 cm X 0.46 cm) and three short alkylsilica (C,J Spherisorb 5-pm C18(15 mm X 4.6 mm) columns in the same coupling holder; Injection 20 pL of 0.2 mg/pL Pi = phosphatidylinositol in chloroform/methanol (211 v/v); mobile phase acetonitrlle/methanol/phosphoric acid (99/3/1 v/v/v); flow rate 3 mL/min. Peaks: SF = solvent front, Pil, Pi2, and Pi3 = molecular species of phosphatidylinositol.
LPC
140
I
I
4
l
l
1
8
1
12
1
1
16
Time (min)
,.__._.. ....
20
-
I
0
1
2
3
4
5
6
;
number of reverse phase column
Flgure 11. Plot of the reduced plate height versus the number of alkylsilica (C,8) columns coupled in series with the reference silica column: (0) Pg = dioleoylphosphatiidylglycerol; (A)Ps = phosphatidylserine; (M) Pe = dioleoylphosphatldylhanolamlne; (A)Pc = dloleoylphosphatiiylcholine;(0)LPC = oleoyllysophosphatklylcholine.
9 shows the variation of SR versus nc for these compounds. To check the accuracy of this procedure, theoretical values
Figure 12. HPLC of commercial phospholipids with a reference silica column and short alkylsilica columns connected by column switching. The arrowhead indicates the valve at 1 mln 40 s (cs = switching of the column). Conditions: mobile phase acetonitrlle/methanol/phosphorlc acid (99/3/1 v/v/v); flow rate 3 mL/mln; detection at 206 nm; injection 20 pL of Pg = dioleoylphosphatldylglycerol;PI = phosphatidylinositol; Ps = phosphatidylserine; Pe = dloleoylphosphatldylethanolamine; Pc = dloleoylphosphatldylcholine; LPC = oleoyllysophosphatidylcholine; SM = sphingomyelin in chloroform/methanol(2/1 v/v) at 0.01 mg/mL. (A) Detection UV A represents the peaks passed through the short alkykilica (&) column. Peaks: SF = solvent front; Pil, P12, and PI3 = molecular specles of phosphatldyllnositol; Pg. (B) Detectlon UV B represents peaks detected directly at the W t of the silica column. Peaks: SF = solvent front; Pser, Pe, Pc, LPC; SM.
of SR, calculated from the k values of the phospholipids given in Table 11,are also plotted (dashed lines) together with the experimental values. A good correlation is observed. It is now possible to class the phospholipids in two groups: (1) With the predominant adsorption mechanism, a negative slope is found (k’c,8/ksi< 1)(dioleoylphosphatidylcholine(Pc) and oleoyllysophosphatidylcholine(LPC)). (2) With the predominant solvophobicretention mechanism, SR increases with anc (k > k ki) (phosphatidylglycerol, phosphatidylserine, and
:j!H
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
PhorphaUdylglycrroi dioleyl
377
Phosphatidyiserine
4000
1
I
e
400
1ow
200
3000 0 0
2
4
6
8
0
101214
2
4
6
8
101214
amount (pg)
amount ( V g )
2000
PhDIDhitidVleUla~oIaminediolcvl
Phosphatidylcholine dioleyl
3000
1000
8
a 4000
2000
e
! i
d
L
1000
0 0,O
1000
0,l
0,2
0,3
0,4
0.5
0,6
0 10
0
20
30
40
50
60
amount (ug)
amount (Fg)
concentration (mg/ml)
Lyrophosphatidylcholinc dialryl
2000
Sphhgomyeline
I , 8000
’
6000
n
5
t
4000
5000
2WO
4000
0 0
5
10
15
amount (pg)
20
25
0
20
40
60
BO
amount (pg)
Flgure 13. Standard response curves of phospholipids in a columnswttching system. Varying amounts are injected in 20 pL of chioroformlmethanol (211). The area is in arbitrary units.
phosphatidylethanolamine). In addition, this system allows the separation of molecular species of phosphatidylserine,as shown in Figure 7, and phosphatidyliiositol, as presented in Figure 10. In terms of selectivity or retention, mixed phases increase the versatility of the separation, but coupling phases in series produce a strong 1- of the system efficiency with the number of additional columns, as shown in Figure 11. 11. Colum Switching (System 111). A switching system is chosen to analyze specifically the solvent front containing phosphatidylglycerol and phosphatidyliiositol. This mixture passes through both stationary phases, silica and alkylsilica. The other phospholipids, which are more retained in the silica system, are detected directly at the outlet of the silica column. This preserves their efficiency and detection sensitivity, by avoiding further dilution in the alkylsilica part of the system. The chromatograms of the two steps of the switching procedure are shown in Figure 12. The upper view of Figure 12 (A) shows the chromatogram obtained with the bidimensional column configuration: at the switching time, there is an artifact peak (stop flow) followed by two molecular species of phosphatidylinositol and a peak of phosphatidylglycerol. The lower trace presents the front of the sample solvent and the remaining phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin). This chromatogram is similar to those obtained with the single silica column shown in Figure 5.
Calibration Curves of the Phospholipids. Calibration curves of the phospholipids were determined in the columnswitching system. Standard curves of phospholipids are given in Figure 13. They show a linear response for amounts
3000
2000
1000
0 0,O
0,l
0,2
0.3
0,4
0,5
0.6
concentration (mg/ml) Flgure 14. Standard curves of phosphatidylinositol in a columnswltchlng system. Variable amounts are injected in 20 pL of chioroforme/methanol(2/1). (A) (El) Pil, (+) Pi2, and (M) Pi3 = molecular species of phosphatidylinositol. (B) Peak area of Pi1 Pi2 Pi3. The area scale is as in Figure 13.
+ +
between 0.1 and 15 pg. The absorbance of sphingomyelin is lower, and it can therefore be quantified between 1 and 40 pg. Phosphatidylinositol was also separated in molecular species, and calibration for these different species and the total peak area is shown in Figure 14. Quantitative Analysis of Pulmonary Surfactant. Pulmonary surfactant was extracted as described in the Experimental Section. Dry surfactant (20 mg) was dissolved in 2 mL of chloroform/methanol (2/1). A 20-pL aliquot of sample was injected into system I (reference silica column) and system ID (column switching). These chromatxgrams are presented in Figure 15. The chromatographic profie obtained with system I (Figure 15A) is similar to the one of Figure 5. This permits the detection of phospholipid classes phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin. With systems I and 111, detection of phospholipid classes (Figure 15B1) are virtually identical. With system 11, however, phosphatidylinositol and phos-
978
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
i
II
I1
I
n
I1
II
/I
rI
I
I
8
1
I
2
%
Flguro 15. HPLC chromatogram of pulmonary surfactant (lnjectlon of 20 CCl of surfactant phospholipid extract (seematerials and method)) (A) wlth referencesilica cdwnn. condtkns: spherisorb 5-pm Sitka (22 cm X 0.46 cm); roWe phase acetonlbile/methanol/phosphorlcadd (9Q/3/1 v/v/v); Row rate 3 Wmh. Peaks: SF solvent front, Pi = phosphatkIyhsM, Ps = pt”t#Ylserine, Pe = d b l e o ~ ~ n o i a d n e , Pc dkleoylphosphatidylchdholine, SM = sphingomyelin. (B)HPLC with reference SHlCa column and a short alkylslHca (C18)column connected by column swltchlng. The arrowhead Indicates the valve at 1 mln 40 s (cs = swltchlng of the column). Conditions: mobile phase acetonitrib/“oi/pho@ork acid (99/3/1 v/v/v); Row rate 3 Wmln. (BI) Detectkn UV B peaks detected directly at the outlet of slllca column. peeks: SF =solvent front; Ps = phosphatidylserine, Pe = dkleoylphosphetklyle,Pc = d k , SM = Sphlngomym. (82) Detection W A peaks detected after separation in the coupled system, passed through the short alkylslllca (C18)column. Peaks: SF = solvent front; Pil, Pi2 = molecular s p e c k of phosphatklyllnosltol; Pg = dioleoylphosphatidyiglycerol.
Table 111. Surfadant Composition As Determined by Column-Switching System (Compared to Data Obtained by DethlofIl“)
phoepholipids pg Pi PS
Pe Pc
SM
% composition of total column switching
*
5.0 0.3 4.60 f 0.06 1.40 i 0.13 19.0 k 0.3 66 0.4 4 0.2
*
mean sd determined by Dethloff
*
19.8 0.3 3.8 k 0.7 0.8 i 0.2 6.4 i 0.6 68.4 0.3 nd
pbatidylglycerol (Figure 15B2)are selectively retained, maicing their quantitative determinationpossible. The quantification of phospholipids in the surfactant was performed by the calibration curves of each solute in the column-switching system. The reaulta of the quantitative analysis are reported in Table III and compared with thw obtained by Dethloff.B
DISCUSSION Selective Retention (SR). As the phospholipids are amphiphilic molecules, they can be retained by both types of interaction, adsorption and solvophobic mechanism. The selective retention, as expressed in eqs 6 and 7, describes the relative contribution of the two stationary phases employed in mixed-phase chromatography. The effect on retention of the solvophobic mechanism depends on the ratio a of the two types of stationary phases as described by eq 3,as well as the capacity factors of the solutes in the two pure phase systems. The cases of dioleoylphosphatidylglycerol (Pg) and oleoyllysophosphatidylcholine (LPC) illustrate well the inversion of selectivity possible in such a system: on the silica column, the capacity factors are 0.18 and 13.4,respectively (Table I). Their elution characteristics in an alkylsilica system with the same mobile phase
lead to capacity factors of 17 and -0.01, respectively (Table
In. Thus for a given mobile-phase composition, optimization of the selectivity of different adjacent peaks can be reached by calculating the optimal stationary-phase ratio. Application of the “SR”Concept to the Optimization of Phospholipid Separations, System 11,coupling in series one silica column with several short alkylsilica columns, permits modification of the composition of the stationary phase (silica/alkylsilica). The SR parameter can be used to optimize the ratio a of these stationary phases as presented in Figure 7. However, a loss of the efficiency with the number of short columns added was observed, especially for phosphatidylserine (the reduced plate height incrmes from 25 to 120 when seven columns are added). This loss of efficiency could be kept small by using a single additional column of equivalent stationary-phase volume. In terms of selectivity optimization, the major goal of the mixed-phase system is to achieve the separation of unresolved peaks. In this system, the phospholipids that are retained and well resolved in the silica column present also a low “solvophobicmechanic"'; for a given mobile phase it appears that some phospholipids present an exclusive “adsorption mechanism” (LPC; others, an exclusive “solvophobic mechanism” (Pg)), as shown in Table 11. For compounds like Ps, Pc, and Pe, well retained by adsorption, the solvophobic effect on the retention cannot be negligible. In the chromatographic systems presented here, the phospholipids studied appear to be retained by one of the two mechanisms or both. The practical advantage of the mixed stationary phases is represented by the resolution of Pg and Pi from the solvent front with a good selectivity. Figure 9 shows the strong “selective” effect of the alkylsilica (C18) column on the retention of Pg compared with a nonretained solvent front (bold line); only one short additional column is enough to strongly improve the resolution of Pg from the solvent front; with three
Anal. Chem. 1992, 64, 379-386
additional columns, molecular species of Pi are resolved. The results obtained with the mixed phases can be improved by the use of the switching system, in this case the separation of Pg from the solvent front can be achieved with a retention time lower than 4 min. This column-switching system keeps the advantages of the single silica column (high efficiency) for most solutes and resolves the remaining complex solvent front (chloroform, phosphatidylglycerol, phosphatidylinositol). Rapid determination in less than 6 min of phosphatidylglycerol and phosphatidylinositolwas carried out (Figure 12). Simultaneously, the other phospholipids are isocratically eluted with a complete run time of 18 min and a sensitivity higher than in a gradient mobile-phase elution. Application of the Coupled System: Separation of Pulmonary Surfactant Analysis. The separation of phospholipids with the switching device is applied to the analysis of pulmonary ox surfactant. The compositions of phosphatidylglycerol (5% ) and phosphatidylethanolamine (15%) have been determined and differ from the data obtained by DethlofP3 for a material of equivalent origin (phosphatidylglycerol = 15% and phosphatidylethanolamine = 5%). With the chromatographic system used in this report, an increased resolution for phosphatidylglycerol and other phospholipids is achieved and permits a better accuracy in the quantitative analysis. In addition, the separation of phosphatidylinositol molecular species gives a new chromatographic profile of the lung surfactant. An automated version of the switching system will allow the elution of all phospholipid classes by heart cut techniques with different mobile phases. It could be suitable for a routine analysis of phospholipids especially to investigate fetal pulmonary maturity in the amniotic fluid.
ACKNOWLEDGMENT We are indebted to Martin Czok and Emmanuelle Brialix for helpful discussions and technical assistance. REFERENCES (1) Denlzot, A. 6.;Tchoreloff, P. C.; Bonanno, L. M.;Proust, J. E.;Lindenbaum, A,; Dehan, M.; Pulsieux. F. Med. Sei. 1991. 7, 37-42.
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RECEIVED for review April 17, 1991. Accepted October 31, 1991.
Counter-Propagation Neural Networks in the Modeling and Prediction of Kovats Indices for Substituted Phenols Keith L. Peterson Division of Science and Mathematics, Wesleyan College, Macon, Georgia 31297 Counter-propagation neural networks are applied to the problem of modeling and predicting the Kovats indices of a set of substituted phenols from their nonempirical structural descriptors. The results are compared to those obtained from quantitative structure-chromatographk retention relationships in the form of multivariate linear regression equations. I find that the neural networks are signtficantiy better at modeling the data, typically giving root mean square errors in Kovats indices between 0 and 10, whereas linear regression equations typically give root mean square errors between 50 and 150. The predictions of Kovats indices with neural networks are better than predictions from regression equatlons by a factor of about 1.25 when the correlatbn coefficient between the structural descriptors and retention index is low. However, when the correiatbn coefficient is high, the regression predlctlons are better than the neural network predictlons by factors between 1.5 and 2.0.
I. INTRODUCTION There have been many attempts at obtaining quantitative structurechromatographic retention relationships (QSRR's) for given classes of compounds.' Typically, either empirical physicochemical parameters or nonempirical structural descriptor parameters have been used in order to obtain quantitative or semiquantitative relationships which allow the prediction of the retention behavior of an individual solute of a given class. The QSRRs are often expressed in the form of a linear equation whose independent variables are the parameters mentioned above and whose dependent variable is a Kovats index. The equation is obtained by performing a multivariate linear regression on data for compounds of a given class on a particular stationary phase. In this paper the use of counter-propagation neural networks as an alternative to linear multivariate QSRRs will be introduced. The usefulness of these neural networks in the
0003-2700/92/0384-0379$03.00/00 1992 American Chemical Society
