A method for the simultaneous determination of alkylacylglycerol, diacylglycerol, monoalkylglycerol, monoacylglycerol, and cholesterol by high-performance liquid chromatography

ANALYTICAL

198,302-307

BIOCHEMISTRY

(1991)

A Method for the Simultaneous Determination of Alkylacylglycerol, Diacylglycerol, Monoalkylglycerol, Monoacylglycerol, and Cholesterol by High-Performance Liquid Chromatography’ Thomas

R. Warne

and Mitchell

Robinson2

Department of Biochemistry, JamesH. Quilkn Collegeof Medicine, East TennesseeState University, Johnson City, Tennessee37614

Received

December

26, 1990

Diradyl- and monoradylglycerols serve as intermediates in the biosynthesis and degradation of glycerolipids. Recently, there has been considerable interest in the role of diradylglycerols as intracellular second mes-

sengers. Both the alkylacylglycerol (AAG)3 and diacylglycerol (DAG) subclasses of diradylglycerols function as activators of protein kinase C following stimulus-induced generation from the hydrolysis of phospholipids (1,2). Monoradylglycerols serve as precursors to, or degradation products of, diradylglycerols (3) and may mediate specific biological activities. For example, monoalkylglycerols (AG) enhance mouse macrophage activation (4) and monoacylglycerols (MG) stimulate insulin secretion in pancreatic islets (5,6). The accumulating evidence supporting the role of diradyl- and monoradylglycerols as biological mediators emphasizes the need for their accurate quantitation. Total diradylglycerols may be analyzed as [32P]phosphatidic acid after conversion with Escherichiu coli DAG kinase and [32P]ATP (7). Selective destruction of DAG fraction by Rhizopus lipase (8) or alkaline hydrolysis (9) permits the separate analysis of AAG alone. AAG and DAG have been quantified separately by staining and densitometry after separation by TLC (10). Several techniques have been developed for the HPLC analysis of chromatophoric derivatives of diradylglycerols prepared from phospholipids (11). Similarly, molecular species of native diradylglycerols have been quantitatively analyzed by reversed-phase HPLC as their benzoate derivatives (12). Normal-phase HPLC analysis of benzoate derivatives of endogenous DAG and AAG have been used to measure total amounts of these diradylglycerols in human neutrophils (13). We describe a method for the quantitative HPLC analysis of the individual subclasses (1-acyl and l-O-al-

’ This work was supported Grant DK38660. * To whom correspondence

3 Abbreviations used: AAG, alkylacylglycerol; glycerol; DAG, diacylglycerol; MDCK, Madin-Darby MG, monoacylglycerok MTBE, methyl tert-butyl

We describe a method for the quantitative analysis of the individual subclasses (1-O-alkyl and 1-acyl) of diradylglycerols and monoradylglycerols. These lipids, along with cholesterol, were separated from other neutral and polar lipids on silica columns and analyzed by normal-phase high-performance liquid chromatography (HPLC) as their benzoate derivatives. Cholesterylbenzoate, alkylacylglycerolbenzoate, diacylglycerolbenzoate, monoalkylglyceroldibenzoate, and monoacylglyceroldibenzoate eluted from HPLC in five distinct zones. The derivatives of diradylglycerols and monoradylglycerols were further separated within each discrete zone on the basis of the total number of aliphatic carbons at the an-l and m-2 positions. Radiolabeled cholesterol and dihexadecanoylglycerol were used to monitor recovery. Amounts of synthetic alkylacylglycerol, diacylglycerol, monoalkylglycerol, and monoacylglycerol as low as 0.2 nmol per subclass could be accurately quantified. The technique was used to determine the content of diradylglycerol and monoradylglycerol subclasses in Madin-Darby canine kidney and CFTL-12 mast cells. This method should prove useful for the quantitation of lipid second messengers in cultUred

302

C&3.

Q 1991

Academic

Press.

by United should

Inc.

States

Public

be addressed.

Health

Service

AG, monoalkylcanine kidney; ether. 0003-2697/91

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

HPLC

ANALYSIS

OF

DIRADYLGLYCEROL

kyl) of diradyl- and monoradylglycerols that has several advantages over previously described methods. The method avoids TLC preparation and associated problems such as isomerization and reduced recovery. Quantitative data may be readily normalized to cholesterol, avoiding additional analyses of protein or lipid phosphorus. Also, the benzoate derivatives may be further analyzed for molecular species composition using reversedphase HPLC. MATERIALS

Methanol, chloroform, hexane, methyl tert-butyl ether (MTBE), and cyclopentane were HPLC grade from Burdick and Jackson (Muskegon, MI). Benzene, benzoic anhydride, and 4dimethylaminopyridine were from Aldrich Chemical Co. (Milwaukee, WI). Prepacked silica columns (PrepSep-Si, 300 mg silica) were obtained from Fisher Scientific (Pittsburgh, PA). All glassware was rinsed twice with chloroform/methanol (2/l, v/v) and air dried prior to use. [1,2,-3H(N)]Cholesterol (62.0 Ci/mmol) was obtained from New England Nuclear (Boston, MA). [3H]Dihexadecanoylsn-glycerol was prepared from L-cr-dipalmitoyl[2-9,103H(N)]phosphatidylcholine (50.0 Ci/mmol, NEN) by phospholipase C (Bacillus cereus, Type XIII, Sigma Chemical Co., St. Louis, MO) hydrolysis with the method of Mavis et al. (14). 1-Monoacyl-rut-glycerol and 1,3diacylglycerol standards were purchased from Sigma. 1,2-Diacyl-sn-glycerol standards were purchased from Sigma or prepared by phospholipase C hydrolysis of the corresponding phosphatidylcholine species (Sigma) as described above. 1-O-Hexadecyl-rucglycerol and 1-0-octadecyl-rut-glycerol were purchased from Sigma and monooleoyl glycerol ether was purchased from Serdary Research Laboratories (London, Ontario). Alkylacylglycerol standards were synthesized by partial acylation of monoalkylglycerol with the respective anhydride (Nu-Chek Prep., Elysian, MN) by the method of Gupta et al. (15) using approximately equal molar amounts of the anhydride and alkylglycerol. The 1-O-alkyl-2-acyl-rut-glycerol product was isolated by TLC using silica gel H plates (Analtech Inc., Newark, DE) developed in hexane/diethylether (40/60, v/v). METHODS

Cell culture and extraction of cell lipids. MadinDarby canine kidney (MDCK) cells (American Type Culture Collection, Rockville, MD) were cultured at 37”C, 5% CO, in Dulbecco’s modified Eagle’s medium supplemented with heat-inactivated fetal bovine serum (lo%, v/v), penicillin (100 U/ml), and streptomycin (100 pg/ml). MDCK cells were passaged by trypsinization and harvested at or near confluence (ca. 1.7 X lo5 cells/ cm2). The interleukin-3-dependent mast cell line,

AND

MONORADYLGLYCEROL

303

CFTL-12, was a gift from Dr. J. Pierce (NIH, Bethesda, MD). The cells were cultured at 37”C, 5% CO, in RPM1 1640 supplemented with heat inactivated fetal bovine serum (lo%, v/v), penicillin (100 U/ml), streptomycin (100 pg/ml), and a supernatant (10% v/v) prepared from the culture media of WEHI-3B cells as a source of interleukin-3. CFTL-12 cells were passaged by dilution and harvested after 4-6 days of culture (ca. 8.0 X lo5 cells/ml). After rinsing twice with 10 ml of respective serum-free media, cells were killed by addition of methanol. [3H]Cholesterol (0.35 PCi) and [3H]dihexadecanoylglycerol (0.08 &i) were added and the lipids were extracted by the method of Bligh and Dyer (16) and processed immediately or stored under nitrogen at -20°C in 0.5 ml of hexane/MTBE (96/4, v/v). Isolation and derivutization of cholesterol, uZkyZucyZgZyceroZ, diacylglycerol, monouZkyZgZyceroZ,and monoacylglycerol. Prepacked silica columns were mounted in a vacuum manifold and serially conditioned with 10 ml isopropanol, 5 ml MTBE, and 15 ml hexane. Lipids from cells or standards were evaporated with nitrogen and transferred to the column by three sequential rinses of 0.5 ml of hexane/MTBE (96/4, v/v). The following solvents were used in a modification of the method of Hamilton and Comai (17) to elute three fractions: (1) 6 ml hexane/MTBE (96/4, v/v) followed by 6 ml hexane/ MTBE/acetic acid (100/2.0/0.2); (2) 6 ml MTBE/acetic acid (10010.2, v/v); (3) 10 ml methanol. Fractions were monitored for cross-contamination by TLC on silica gel H plates developed in hexanejdiethylether (40/60, v/v) and lipids components visualized by spraying to saturation with 10% cupric sulfate in 10% phosphoric acid and heating to 250°C. Prepacked silica columns were routinely reused following washing with isopropanol, MTBE, and hexane, heated at 75°C for 16 h and stored desiccated or at 75°C. Samples from cells (Fraction 2) or standards of cholesterol and diradyl- and monoradylglycerols were evaporated with nitrogen and converted to the benzoate derivatives (18) using 1 mg benzoic anhydride and 0.4 mg 4dimethylaminopyridine in a total volume of 120 ~1 benzene. The samples were incubated under nitrogen for 3 h at 25°C and the reaction was terminated by the addition of 200 ~1 ammonium hydroxide followed by incubation for 15 min at 25°C. The lipids were extracted by adding 2.5 ml acetonitrile and 0.5 ml H,O followed by 3.0 ml hexane. After centrifugation, the upper hexane layer was recovered and the lower layer was reextracted twice with 3.0 ml hexane. HPLC unczZysis. HPLC was performed using a Perkin-Elmer Series 410 LC Pump and LC-95 uv-Visible LC spectrophotometer. For data collection, the detector was interfaced to Maxima 820 Chromatography Workstation on an NEC APCIV computer. Lipid components were separated with a Beckman Ultrasphere-Si column (4.6 mm X 25 cm, 5-grn pore size) using an isocratic

304

WARNE

AND

mobile phase of cyclopentane/MTBE (97/3, v/v) at 1 ml/min and 35°C. All samples were dissolved in the mobile-phase solvent for injection. The column wasperioditally washed with isopropanol/cyclopentane/MTBE (50/48.5/1.5, v/v/v) to remove accumulated reagents. For determination of retention time of individual molecular species, benzoate derivatives were directly prepared from cholesterol and respective molecular species of AAG, DAG, AG, and MG. Determination of mass quantities of benzoate derivatives eluting from the HPLC was based on a molar absorptivity at 230 nm of 13,200 M-’ cm-’ for benzoyl esters (18). For the conditions of this study (l-cm detector path length with a response of 1 V per absorbance unit and a flow rate of 1.0 ml/min), this corresponds to an integrated peak area of 792,000 PV * s * nmol-’ for monobenzoylated derivative and 1,584,OOO PV * s * nmol-’ for dibenzoylated compounds. The response factor of the instrument was verified to be within 10% of this value by injection of known quantities of cholesterylbenzoate and also by determination of the response per mass quantity of 1,2-[3H]dipalmitoylglycerobenzoate prepared from 1,2-[3H] dipalmitoylglycerophosphatidylcholine of known specific radioactivity. RESULTS

AND

DISCUSSION

Cholesterol and diradyland monoradylglycerols were efficiently separated from other neutral and polar lipids on silica columns using a modification of the method of Hamilton and Comai (17). When monitored by TLC charring, no cross-contamination was evident in fractions that contained triglycerides, cholesterol esters, and free fatty acids (Fraction 1); cholesterol, diradylglycerols, and monoradylglycerols (Fraction 2); and polar lipids (Fraction 3). In addition, greater than 98% of the radioactivity of [3H]cholesterol and [3H]dihexadecanoylglycerol eluted in Fraction 2. The components in Fraction 2 were reacted with benzoic anhydride in the presence of DMAP (19) to yield derivatives of cholesterol (cholesterylbenzoate), monoradylglycerols (monoradylglycerodibenzoates), and diradylglycerols (diradylglycerobenzoates). The amounts of benzoylation reagents were adjusted to minimize contaminating HPLC peaks associated with the reagents while assuring complete (>98%) derivatization of total amounts of cholesterol, AAG, DAG, AG, and MG commonly encountered in cell preparations, i.e., approximately 500 nmol. We determined retention times of benzoate derivatives of cholesterol and known representative molecular species of AAG, DAG, AG, and MG (Fig. 1, Table 1). The benzoate derivatives separated into five discrete zones. Within each zone, saturated molecular species eluted according to the total number of aliphatic carbons at the sn-1 and sn-2 positions and showed a linear

ROBINSON

0

” 10

3

s

”

3

’

15

RETENTION

FIG. 1. Representative tives of known molecular glycerols. Peak numbers

*

”

”

”

20

TIME

” 25

,I*

’ 30

(min)

HPLC chromatograms of benzoate derivaspecies of monoradylglycerols and diradylcorrespond to those listed in Table 1.

relationship with log relative retention time (Fig 2). For DAG, the benzoate derivatives of 1,3 isomers showed a consistent decrease in retention time from that of the corresponding 1,2 isomers (Fig. 1, Fig. 2). The regular pattern of separation should enable preliminary identification of component peaks of diradyl- and monoradylglyceroi subclasses. However, the complex mixtures of species present in biological samples will likely contain overlapping peaks within each subclass. Another diradylglycerol subclass, alk-1-enylacylglycerol, elutes separately from other subclasses immediately before AAG (data not shown). However, this region also contains unknown contaminants. Although the method was developed to measure the quantities of the benzoylated derivatives of diradyl- and monoradylglycerol subclasses relative to the amounts of cell cholesterol, absolute quantities of these components can be measured directly from integrated peak areas and corrected for recovery using radiolabeled cholesterol and dihexadecanoyl-sn-glycerol. In order to confirm absolute quantitation, we analyzed known amounts of cholesterol (50-250 nmol) and molecular species of AAG, DAG, AG, and MG (0.05-7.5 nmol per subclass). As shown in Fig. 3, the assay was highly linear (r2 = 0.99, slope = 0.98) when the mass of diradyl- and monoradylglycerols analyzed was at least 0.2 nmol per subclass. The recovery of total [3H]cholesterol radioactivity in the cholesterylbenzoate peak was consistently similar to the recovery of added [3H] dihexadecanoylsn-glycerol eluting in the diacylglycerobenzoate area. Although these results indicate that absolute quantitation of the four subclasses and cholesterol can be achieved, such determinations depend upon accurate evaluation of the HPLC detector response per mass of eluting benzoyl derivative. Although we found that the response

HPLC

ANALYSIS

TABLE Retention

Times

acylglycerolbenzoates benzoates Peak number“

of

DIRADYLGLYCEROL

1

Molecular

AlkylMonoalkcylglyceroldi-

Species

and Monoacyl-,

Molecular

OF

species6

in

Diacyl-,

Retention

time’

(min)

Cholesterol Alkylacylglycerol 2

3 4 5 6 Monoalkylglycerol 7 8 9 Diacylglycerol 10 11

12 13 14 15 16 17 18 19 20

21 22 Monoacylglycerol 23 24 25

l&O-16:0 l&O-14:o l&O-14:0 l&O-18:1 l&-18:1 16:0-l&1

8.68 8.98 9.28 8.37 8.57 8.99 11.46 11.86 11.84

l&O 16:0 l&l l&O-18:0 l&O-19:0 l&O-16:0 15:0-15:o 14:0-14:o 13:0-13:o l&O-18:0 16:0-16:0 14:0-14:o l&l-l&O 16:0-l&1 l&l-160 l&O-20:4 l&l-18:l l&O-18:2 l&2-18:2 l&O 16:0 14:o

(1,3) (1,3) (1,3)

18.33 18.71 19.68 20.53 21.30 22.49 16.91 18.39 19.99 18.52 19.25 18.73 18.93 20.05 20.05 21.26 26.29 27.33 28.54

a Peak numbers correspond to those shown in Figs. 1 and 2. * Expressed as total number of aliphatic carbons:total number of double bonds. ’ Median number of determinations was 5. The standard deviation was less than 3.75% of the mean retention time. d Since the cholesterol peak was routinely off scale, the window of retention time is given.

AND

AAG, DAG, AG, and MG subclasses fell into distinct zones defined by the corresponding synthetic standards. Approximately 2 X lo7 MDCK or CFTL-12 cells were required to provide sufficient material for reproducible quantitation of the low levels of AAG, DAG, AG, and MG (Table 2). Analysis of this amount of total lipid resulted in an offscale cholesterylbenzoate peak, precluding direct measurement of cholesterol mass. The cholesterylbenzoate peak (R, = 3:10-4:lO) was therefore collected and l-2% reinjected. The cholesterol content of the original sample was determined from the resulting peak area and radioactivity. Zones of elution for AAG, DAG, AG, and MG that correspond to representative molecular species are given in Fig. 4C. Unidentified or contaminant peaks did not interfere with the well-defined areas of elution. The region between AG and DAG typically contained peaks that appear to be associated with the benzoylation reagents. In addition, a peak found intermediate between DAG and MG (Figs. 4A and 4B, peak X) is likely an unidentified cellular component. In order to confirm that endogenous contaminants were not present in the regions identified as AAG and DAG, the benzoate derivatives of MDCK cell lipids eluting in these zones were collected and analyzed by reversed-phase HPLC (12). The pattern of molecular species was similar to that previously reported for diradylglycerobenzoates prepared from purified MDCK cell AAG and DAG (12) and extraneous peaks were not observed. Although our analyses indicate that the peaks eluting in the zones defined as benzoyl derivatives of AAG, AG, DAG, and MG are free of unidentified or interfering components for the particular cells we have examined, analyses of other tissues may require positive identification of the peaks grouped in these zones by reversed-phase HPLC. The method we describe for the quantitative analysis of cholesterol, AAG, DAG, AG, and MG has several po-

35 is 0

factor corresponded to the standard value for the molar absorptivity of benzoyl derivatives, this value depends on parameters such as the flow rate and the path length of the detector and should be experimentally verified for each instrument. Alternatively, quantities of the diradylglycerols and monoradylglycerols can be normalized to cholesterol. Since the same chromatophore is used to measure each compound, results expressed in this way are not dependent on the response factor of the detector. Figure 4 shows the HPLC separation of benzoate derivatives of cholesterol, DAG, AAG, MG, and AG from MDCK and CFTL-12 cells. Peaks corresponding to the

305

MONORADYLGLYCEROL

g30s

11

17

12

3

13 18

0 5

25

2 i a

2.

d z

10 “s3’9tG DAG

1 AAG -1 _ -2

11'

14 15

ii

_

-

AG

-

MG

7 81

15 1 8

I I 9 10

-

23 24 I

I I I I III

RETENTION

TIME

I I II,, 20

-

25 \I,,

30

(min)

FIG. 2. Correlation between log retention time and aliphatic carbon number of benzoate derivatives of monoradylglycerol and diradylglycerol. Peak numbers correspond to those listed in Table 1.

306

WARNE

AND

ROBINSON

TABLE Analysis

2

of the Contents of Cholesterol and and Diradylglycerols in MDCK

Subclasses of Monoradylglycerols and CFTL-12 Cells

Diradylglycerol Cell

line

Cholesterol

AAG nmol

MDCK”

194.3 f 9.7

per 100 nmol

1.19 + 0.06

92.6 k 4.8

-

per 100 nmol

for AAG,

3.42 f 0.08

1.21 f 0.06

0.16 + 0.01

0.65 + 0.05

0.08 t 0.02

1.59 + 0.14

1.22 + 0.08

1.69 + 0.61

1.46 + 0.74

cholesterol

2.62 + 0.15

3.12 + 0.81

Note. All values are expressed as means + SE. ’ On the basis of four separate determinations. * On the basis of six separate determinations, except

MG

per lo7 cells

3.49 k 0.42 nmol

AG

2.04 + 0.09

nmol CFTL-12*

DAG per dish

2.31 f 0.17 nmol

-

Monoradylglycerol

cholesterol 2.91 f 0.85

which

tential advantages. The lipid components of interest are isolated from total lipid extracts in a single purification step. The use of prepacked silica columns avoids TLC and the problems associated with the visualization, isomerization, and recovery of the low levels of diradyland monoradylglycerols found in most tissues. The derivatives are separated from the bulk of the reagents by

was based

on three

separate

determinations.

a simple extraction and can be directly applied to HPLC without further purification. Quantities of AAG, DAG, AG, and MG can be readily normalized to cholesterol

t z

0.10

i? 8 z

0.16

E

0.16

Lk

0.14

E

0.12 0.10

FIG. 3.

Quantitation of known amounts of cholesterol and selected molecular species of monoradylglycerol and diradylglycerol. Regression line, y = r-0.98 + 0.01, P = 0.99. The following lipid standards were used: cholesterol, l-0-octadecyl-ret-glycerol, l-Ohexadecyl-rat-glycerol, 1-octadecanoyl-rot-glycerol, l-hexadecanoylrat-glycerol, 1-0-octadecyl-2(3)tetradecanoyl-rat-glycerol, l,%-dioctadecanoyl-sn-glycerol, 1,2-dihexadecanoyl-an-glycerol, 1,2-ditetradecanoyl-sn-glycerol, l-tetradecanoyl-rut-glycerol.

0

5

10

15

RETENTION

FIG. 4.

20

25

TIME

(min)

30

35

Representative HPLC separations of benzoate derivatives of cholesterol, monoradylglycerol, and diradylglycerol subclasses from (A) MDCK, (B) CFTL-12 cells, and (C) retention time standard. Peak identification for individual DAG, AAG, MG, and AG molecular species shown in (C) is given in Fig. 1.

HPLC

ANALYSIS

OF DIRADYLGLYCEROL

and, thus, preclude additional analyses of protein or phosphorus. The diradyl- and monoradylglycerol subclasses can be collected and directly analyzed at the level of individual molecular species by reversed-phase HPLC (X&20). Also, the method measures both 1,2 and 1,3 isomers of AAG and DAG, unlike procedures that utilize enzymatic conversion of diradylglycerols to phosphatidate (7-9). The results of analyses of the diradylglycerol and monoradylglycerol subclasses in CFTL12 and MDCK cell lines shows that the technique is applicable to the study of lipid second messengers in cultured cells. REFERENCES 1. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U., and Nishizuka, Y. (1980) J. Biol. Chem. 255,2213-2276. 2. Ford, D. A., Miyake, R., Glaser, P. E., and Gross, R. W. (1989) J. Bill. Chem. 264,13,818-13,824. 3. Hata, Y., Ogata, E., and Kojima, I. (1989) Biochem. J. 262,947952. 4. Yamamoto, N., St. Clair, D. A., Homma, S., and Ngwenya, B. Z. (1988) Cancer Res. 48,6044-6049. 5. Zawalich, W. S., Zawalich, K. C., and Rasmussen, H. (1989) Diebetologia

32,360-364.

6. Zawalich, W. S., and Zawalich, K. C. (1990) Mol. Cell. Endocrinol. 68,129-136.

AND MONORADYLGLYCEROL Preiss, J., Loomis, R. C., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261,8597-8600. 8. Tyagi, S. R., Burnham, D. N., and Lambeth, J. D. (1989) J. Biol. Chem.

264,12,977-12,982.

9. Rider, L. G., Dougherty, mu&.

R. W., and Niedel, J. E. (1988) J. Zm-

140,200-207.

10. Agwu, D. E., McPhail, Biophys.

L. C., and Wykle, R. L. (1989) Biochem.

Res. Commun.

169,79-86.

11. Patton, G. M., and Robins, S. J. (1990) in Methods in Enzymology (Murphy, R. C., and Fitzpatrick, F. A., Eds.), Vol. 187, pp. 195-215, Academic Press, San Diego. 12. Warne, T. R., and Robinson, M. (1990) Lipids 25,748-752. 13. Agwu, D. E., McPhail, L. C., Chabot, M. C., Daniel, L. W., Wykle, R. L., and McCall, C. E. (1989) J. Bill. Chem. 264,1405-1413. 14. Mavis, R. D., Bell, R. M., and Vagelos, P. R. (1972) J. Biol, Chem. 247,2835-2841. 15. Gupta, C. M., Radhakrishnan, R., and Khorana, H. G. (1977) Z’roc. Natl.

Acad.

Sci. USA

74,4315-4319.

18.

Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 627-643. 17. Hamilton, J. G., and Comai, K. (1988) Lipids 23,1150-1153. 18. Sakai, K., Taniguchi, T., Shimomura, R., Asahi, M., Kobayashi, T., Inazu, T., Nakamura, S., and Yamamura, H. (1989) Biochem. Biophys.

Res. Commun.

165,680-684.

19. Blank, M. L., Cress, E. A., Lee, T-C., Stephens, N., Piantadosi, C., and Snyder, F. (1983) Anal. B&hem. 133,430-436. 20. Blank, M. L., Robinson, M., Fitzgerald, V., and Snyder, F. (1984) J. Chromatogr.

296,473-482.